Mechanistic Multi-Enzyme Engineering for High-Yield Bilirubin Biosynthesis

Mechanistic Multi-Enzyme Engineering for High-Yield Bilirubin Biosynthesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mechanistic Multi-Enzyme Engineering for High-Yield Bilirubin Biosynthesis Rongzhen Zhang, Zhentao Jiang, Jingxin Rao, Caokai Zhu, Yamiao Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6554107/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Bilirubin biosynthesis has long been limited by low yields and unclear bottlenecks. Here, we report a fully in vitro pathway that coverts heme to bilirubin with the highest reported titer. Through systematically screening and engineering, we identified two hidden challenges: Fe²⁺ causes intermediate degradation, and CO inhibits heme oxygenase activity. Initial yields stalled at 48.1% due to Fe²⁺-induced biliverdin and bilirubin breakdown. We revealed that Fe²⁺ interacts with deprotonated biliverdin and bilirubin, triggering oxidative ring-opening degradation via O₂-mediated radical mechanism. DFT calculations showed Fe²⁺-ligand complexes reduce the HOMO-LUMO gap, enhancing their elector transfer susceptibility. Competitive chelation of Fe 2+ and protonation-modulation boosted yield to 80.1%. Furthermore, heme-CO complexes block O 2 -activation for accessing heme oxygenase. Introducing carbon monoxide dehydrogenase for CO removal and formate dehydrogenase for NADPH-recycling enabled efficient bilirubin synthesis of 1.7 g/L and 95.8% yield—a 20-fold improvement. Our work shows byproducts control is the key to stabilize heme-related pathways and as an advanced tool in synthetic biology. Biological sciences/Biotechnology/Industrial microbiology Biological sciences/Biochemistry/Biocatalysis Biological sciences/Biochemistry/Enzymes/Multienzyme complexes Biological sciences/Chemical biology/Computational chemistry Bilirubin multi-enzyme cascade radical attack metal coordination carbon monoxide inhibition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Bilirubin (BR), the principal bile pigment, was historically considered a metabolic waste product due to its pathological accumulation in conditions such as kernicterus and neonatal jaundice 1-3 . However, recent studies have revealed its beneficial physiological roles. BR possesses potent antioxidant activity, protecting lipids, proteins, and other biomolecules from oxidative stress⁴. Moderate serum BR levels are associated with reduced risks of cardiovascular and metabolic diseases³, and BR exerts neuroprotective effects by mitigating neuronal damage in the central nervous system⁵. Its anti-inflammatory, antioxidant, and antiviral properties have also been investigated in the context of COVID-19 treatment⁶. Furthermore, BR is the primary active compound in Calculus bovis , a traditional Chinese medicine with significant market value in China⁷. Although BR is abundant in animal bile, conventional extraction from porcine or bovine sources is inefficient and cost-prohibitive, yielding only ~300 mg of BR per liter of bile⁸. Synthetic biology approaches have enabled enzymatic and de novo biosynthesis of tetrapyrrole compounds, including BR⁹ , ¹⁰. In nature, BR is derived from heme degradation: heme oxygenase (HO, EC 1.14.14.18) oxidizes heme to biliverdin (BV), which is subsequently reduced to BR by NADPH-dependent biliverdin reductase (BVR, EC 1.3.1.24)¹¹ – ¹⁵ (Fig. 1a). HO isoforms exhibit regioselectivity by cleaving specific methylene bridges on the porphyrin ring to produce BV isomers (IXα–γ), but most BVRs selectively convert BV IXα into BR IXα—the biologically active form referred to hereafter as BR. HO-mediated heme degradation requires three equivalents of O₂ and seven equivalents of NADPH, but HO lacks intrinsic cofactor-binding domains. Instead, electron transfer is facilitated by redox partner proteins (RPs), which typically consist of a large NADPH/FAD-binding domain and a smaller FMN-binding domain¹⁶ – ¹⁸. In some organisms, these domains are distributed across separate proteins and use Fe–S clusters to shuttle electrons¹⁹ – ²¹ (Fig. 1d). RP conformational changes regulate their activity: the closed conformation transfers electrons from NADPH to FMN, while the open conformation enables FMN to reduce the heme-bound HO²² (Fig. 1b). Subsequent O₂ binding and activation trigger porphyrin ring cleavage, generating BV, Fe²⁺, and carbon monoxide (CO) as byproducts (Fig. 1c, Supplementary Fig. 1)¹⁶. BVR is a nicotinamide-dependent oxidoreductase with a Rossmann-fold N-terminal domain²³ , ²⁴. Structural studies have revealed that two BV molecules bind in the BVR active site; the distal BV may serve as an electron relay during the reduction of the proximal BV to BR²⁴ (Fig. 1e and 1f; Supplementary Fig. 2). De novo heme biosynthesis via metabolic engineering has been widely explored²⁵ – ²⁸, and redirection of metabolic flux toward BR using HO and BVR has been attempted²⁹. However, the complexity of the biosynthetic pathway has limited yields, with the highest reported BR titer at just 75.5 mg/L³⁰. Alternatively, a two-step enzymatic conversion of heme to BR holds greater promise. Although diverse HO sources have been explored—including plant³¹, bacterial³², and cyanobacterial³³ enzymes—BV titers remain modest (up to 132 mg/L)³¹, and BVR-mediated BR production has reached only 325 mg/L³⁴. Notably, one-pot enzymatic synthesis of BR from heme has not yet been realized. Importantly, the tetrapyrrolic structures of BV and BR retain metal-chelating properties similar to heme 35 . Transition metal complexes such as BV–Cu²⁺ are prone to oxidative degradation³⁶ , ³⁷, and BR can form Ca²⁺ and Cu²⁺ complexes that generate reactive oxygen species (ROS), leading to its breakdown³⁸ , ³⁹. In particular, Fe²⁺, a byproduct of HO activity, may compromise the structural integrity of both BV and BR. Additionally, CO—another byproduct—has strong ligand-binding properties. Its polarized bond and high electron density at the carbon center allow it to outcompete O₂ for coordination with heme iron⁴⁰. CO binding stabilizes heme–CO complexes, which inhibit the formation of O₂-activated catalytic intermediates, thus preventing substrate access and further enzymatic turnover. These mechanistic insights suggest that uncontrolled byproduct interactions may be a critical factor limiting BR biosynthesis yields, which currently remain in the milligram per liter range. In this study, we constructed a compact enzyme library through phylogenetic analysis to identify high-activity variants of HO, RP, and BVR, which were heterologously expressed in E. coli BL21 (DE3). Truncation of hydrophobic regions improved the soluble expression of HO and RP. To overcome the biosynthetic bottlenecks, we employed computational modeling to elucidate key mechanisms of byproduct interference. Density functional theory (DFT) simulations revealed Fe²⁺-induced O₂ activation and radical-driven oxidative degradation of BV/BR. We also demonstrated that CO-heme binding substantially increases the free energy of heme–CO complexes, inhibiting formation of O₂-coordinated intermediates and blocking substrate access to the enzyme active site. Guided by these insights, we developed an optimized bilirubin biosynthesis system that integrates metal chelation, protonation state control, and enzymatic CO elimination. This strategy enabled gram-scale BR production with the highest yield reported to date, offering a robust platform for industrial-scale synthesis. Results Screening of key enzymes HO, RP, and BVR for construction of the BR biosynthetic pathway Mammals and plants typically harbor two heme oxygenase isoforms—inducible HO-1 and constitutive HO-2⁴¹—which share high sequence and structural similarity, along with a conserved catalytic mechanism. In contrast, microorganisms usually encode a single HO isoform capable of synthesizing BR IXα, while some bacteria express HugZ-family heme-degrading enzymes that generate alternative isomers such as BR IXβ and BR IXδ⁴². Evolutionary divergence is also reflected in the redox partner proteins (RPs) that support HO activity. In animals and fungi, RPs are generally NADPH-cytochrome P450 oxidoreductases (CPRs; EC 1.6.2.4)⁴³. By contrast, bacterial and plant systems utilize two discrete proteins for electron transfer: the first protein—either a ferredoxin (FDX) harboring Fe–S clusters or a flavodoxin containing FMN—accepts electrons from NADPH, and the second—ferredoxin-NADP⁺ reductase or flavodoxin-NADP⁺ reductase (FDR)—completes the transfer 2 ⁰ ,2 ¹. Biliverdin reductase (BVR), which catalyzes the conversion of BV to BR, is broadly distributed among animals and bacteria. In animals, BVRs are classified into BVR-A and BVR-B subfamilies, distinguished by regioselectivity⁴⁴ , ⁴⁵. Bacterial BVRs predominantly generate BR IXα, although some rare variants utilize F₄₂₀H₂ as a cofactor in place of NADPH⁴⁶. To identify the most effective enzyme combinations for BR biosynthesis, we performed phylogenetic analysis of HO (Fig. 2a), RP (Fig. 2b), and BVR (Fig. 2c), selecting evolutionarily diverse candidates to maximize functional variability while maintaining representation across known classes. Representative enzymes were heterologously expressed in E. coli BL21 (DE3) (Supplementary Fig. 3), purified, and subjected to activity screening. For the heme-to-BV conversion step, we tested various HO-RP pairings. Among these, Rattus norvegicus HO-1 ( Rn HO-1) in combination with its native CPR ( Rn CPR) yielded the highest activity, producing 53.2 mg/L BV within 10 minutes (Fig. 2d). For the subsequent BV-to-BR step, the BVR-A also from Rattus norvegicus ( Rn BVR-A, hereafter referred to as Rn BVR) exhibited the most robust catalytic performance, generating 338 mg/L BR in 10 minutes (Fig. 2e). Terminal truncation of HO and CPR enhances soluble expression without compromising catalytic efficiency Soluble expression of Rn HO-1 and Rn CPR in E. coli was initially limited, consistent with the presence of predicted membrane-associating domains. Structural analysis revealed that the N-terminal residues 1–54 of Rn CPR and the C-terminal residues 268–289 of Rn HO-1 comprise highly hydrophobic regions (Fig. 3a), previously implicated in anchoring these proteins to the endoplasmic reticulum membrane⁴⁷. To enhance expression, we implemented a stepwise truncation strategy to remove these regions. Soluble expression levels increased with the extent of truncation, with the fully truncated variants Rn HO-1 Δ22 and Rn CPR Δ54 exhibiting the highest yields (Fig. 3b). To confirm that truncation did not impair enzyme function, we first optimized the Rn CPR-to- Rn HO-1 ratio for maximal catalytic activity (Supplementary Fig. 4), then evaluated the kinetic properties of the truncated enzyme pairs. All Rn HO-1 and Rn CPR truncation combinations retained comparable heme affinity ( K ₘ) and catalytic efficiency ( k c ₐₜ/ K ₘ) relative to their full-length counterparts (Fig. 3c and Supplementary Fig. 5), indicating that removal of membrane-associated domains did not compromise catalytic function. By contrast, strong substrate inhibition observed in the downstream bilirubin reductase ( Rn BVR) reaction precluded accurate kinetic modeling (Supplementary Fig. 6). Low yield in cascade processes: An unexpected phenomenon Despite the use of cell lysates with equal concentrations of catalysts, the combination of Rn HO-1 Δ22 and Rn CPR Δ54 exhibited a significantly faster reaction rate than the wild-type enzyme combination. However, an unexpected deviation from stoichiometric predictions was observed: while heme consumption and biliverdin (BV) accumulation were expected to be directly proportional, at 80% substrate conversion, BV yield only reached approximately 50%, with BV levels declining sharply as substrate consumption slowed (Fig. 4a). Notably, this issue was absent in the BVR-catalyzed BV-to-BR conversion, where reaction kinetics aligned with expectations (Fig. 4b). The faster reaction rate of Rn HO-1 Δ22 / Rn CPR Δ54 suggested a high catalytic activity for the heme-to-BV step; however, the observed discrepancy in BV yield points to an additional factor influencing the reaction. Given the significantly higher specific activity of Rn BVR (Fig. 3c), which catalyzed the subsequent BV-to-BR conversion, we hypothesized that the slower first step of the cascade (heme-to-BV) might be the bottleneck. To optimize reaction conditions and better balance cascade kinetics, we refined the experimental setup (Supplementary Fig. 7). After optimizing conditions, we performed one-pot BR synthesis from heme. While the conversion rate of heme increased to 83.9% after 150 minutes, the BR yield plateaued at 48.1% (Fig. 4c). This low yield could not be attributed to external factors, such as UV irradiation 48 or ROS 49 (e.g., H₂O₂), which are known to cause degradation of BR into byproducts like propentdyopents, as our reactions were conducted in the dark and without exogenous ROS. In contrast, the Rn BVR-catalyzed reaction alone performed as expected, suggesting that the issue lies in the earlier heme-to-BV conversion step. We hypothesize that byproducts generated during heme-to-BV conversion, such as Fe²⁺ or CO, may destabilize BV or BR, thereby limiting the final BR yield. Unveiling the mechanism of Fe²⁺-enhanced product reactivity and oxidative degradation promotion We first explored the role of Fe²⁺, a byproduct of HO-catalyzed heme degradation, in promoting the reactivity and oxidative degradation of bilirubin (BR) and its intermediate biliverdin (BV). When standard solutions of BV and BR were incubated with Fe²⁺ under enzymatic reaction conditions (35°C, pH 7.5) for 150 minutes, HPLC analysis revealed the emergence of multiple unknown compounds (Fig. 5a and 5b), alongside a significant alteration in the UV-Vis spectra (Supplementary Fig. 8), which corresponded with a reduction in the peak areas of BV and BR. LC-MS analysis identified 13 potential degradation products (Supplementary Figs. 9-11). Given the established coordination chemistry between porphyrins and metal ions, we hypothesized that Fe²⁺ initially coordinates with BV/BR, thereby triggering degradation. DFT-based electrostatic potential surface analysis suggested that deprotonation of the central nitrogen atoms in BV and BR reduces their local electrostatic potential, enhancing Fe²⁺ coordination (Supplementary Fig. 12a). Time-dependent DFT (TD-DFT) calculations further indicated that deprotonation of BV induces red shifts in the Soret (~380 nm) and Q bands (690 nm), with a noticeable amplification in Q-band intensity (Supplementary Fig. 12b and 12c), consistent with the experimental UV-Vis changes observed (Fig. 6c). These findings confirm that Fe²⁺ preferentially interacts with deprotonated BV/BR (denoted as deBV/deBR), aligning with prior studies on porphyrin-metal ion coordination 37 . DFT calculations revealed favorable coordination energies for deBV-Fe²⁺ (-126.5 kcal/mol) and deBR-Fe²⁺ (-133.5 kcal/mol) (Fig. 5c). Notably, upon Fe²⁺ binding, deBR transitions from a saddle-shaped to a distorted planar conformation. LC-MS confirmed Fe²⁺-mediated oxidative degradation of BV and BR without the need for exogenous reactive oxygen species (ROS). We propose that Fe²⁺ acts as a coordination core, recruiting axial ligands such as O₂ or OH⁻ from the solvent, which in turn enhances the reactivity of the entire complex. To explore this hypothesis further, we investigated various possible axial coordination modes (Supplementary Figs. 13 and 14). Among these, deBV-Fe-OH⁻ exhibited the lowest coordination energy, but OH⁻ ligands poorly attack the porphyrin ring. Therefore, we identified plausible coordination modes, including deBV-Fe-O₂-OH (-9.5 kcal/mol) and deBR-Fe-O₂ (-27.6 kcal/mol) (Fig. 5d). Frontier molecular orbital (FMO) analysis revealed that Fe²⁺ coordination minimally alters the HOMO/LUMO distributions on the porphyrin ring (Fig. 5e and 5f). However, axial ligand binding (such as O₂) reduces the LUMO electron density on the porphyrin ring while shifting LUMO localization toward the O₂ ligand. Notably, Fe²⁺ coordination has minimal impact on the overall HOMO-LUMO gap, but the introduction of axial ligands significantly narrows the gap (from 2.07 eV to 0.56 eV for deBV-Fe-O₂-OH and from 2.83 eV to 0.72 eV for deBR-Fe-O₂). These findings suggest that complexes with axial ligands, such as deBV-Fe-O₂-OH and deBR-Fe-O₂, exhibit enhanced reactivity, rendering them energetically favorable for single-electron transfer. Given that FMO analysis indicated O₂ may act as an electron acceptor, and combining with other heme enzyme mediated ring opening reaction mechanisms 50 , we propose that this coordination facilitates the formation of superoxide radicals (O₂ → O₂⁻•), which then trigger a radical cascade leading to meso -carbon attack and subsequent oxidative degradation (Fig. 5g). The proposed mechanism includes the following steps: (1) direct radical addition of O₂⁻• to meso -carbon, generating a Fe-O-O-carbon intermediate with a β-carbon radical; (2) homolytic cleavage of the O-O bond, forming epoxide intermediates and causing Fe-O bond cleavage; (3) hydrogen transfer from meso -carbon to the epoxide ring, resulting in the formation of a meso -carbon radical; and (4) free radical addition of another superoxide radical to meso -carbon, inducing a ring-opening reaction and leading to oxidative degradation products. This mechanism positions Fe²⁺ not just as a passive byproduct of heme degradation, but as an active driver of porphyrin instability, promoting both structural disruption and radical initiation. Preventing Fe²⁺-induced degradation through competitive chelation and protonation state modulation To mitigate Fe²⁺-induced degradation, we tested various chelators. While all chelators preserved the absorption features of BV and BR (Supplementary Fig. 15), tartrate and other chelators inhibited enzyme activity in both HO- and BVR-mediated reactions (Fig. 6a and 6b). Etidronic acid (HEDP) emerged as the most effective chelator, significantly enhancing BV and BR yields by 149.8% and 145.6%, respectively (Fig. 6a and 6b). Acid-base titration and DFT calculations confirmed that HEDP exists as di-/tri-deprotonated species at the enzymatic pH, and coordination titration established a 2:1 ratio of HEDP to Fe²⁺ (Supplementary Fig. 16). Despite this favorable coordination (ΔG = -106.9 kcal/mol, Supplementary Fig. 17), residual degradation products persisted (Fig. 6e and 6f), suggesting that the suppression of deBV/deBR-Fe²⁺ formation was incomplete. Given that Fe²⁺ coordination is pH-dependent, we optimized the pH to further mitigate degradation. While BR precipitated under acidic conditions, BV studies across a pH range of 3.5-9.5 showed progressive red shifts in the Soret and Q bands upon deprotonation (Fig. 6c). Addition of Fe²⁺ under alkaline conditions drastically reduced BV absorption, whereas acidic pH preserved its spectral integrity (Fig. 6d). Adjusting the pH to mildly acidic conditions, in combination with HEDP addition, effectively reduced degradation (Fig. 6e and 6f). The enzyme activity and overall reaction efficiency were optimized by adjusting the HEDP concentration (15 mM) and pH (6.5), ensuring minimal degradation and enhanced product yields (Supplementary Fig. 18). Another cause of unsatisfactory yield in cascade reactions: CO byproduct inhibits heme utilization by HO enzyme Under optimized HEDP dosage and pH conditions to suppress Fe²⁺ interference, the BR yield increased significantly to 80.1% (1953 mg/L, 3 mM heme as substrate) with the Rn HO-1 Δ22 , Rn CPR Δ54 , and Rn BVR mixture. However, approximately 20.5% of heme remained unconverted during the late stages of the reaction (Fig. 7a). This phenomenon led us to consider CO poisoning 51 , a well-documented issue where CO, another byproduct of HO catalysis, may act as an inhibitor. CO is generated from the meso -carbon atom of heme, transported through a narrow gas channel in the enzyme, and subsequently released into the solvent environment (Fig. 7c). DFT calculations revealed that heme-CO coordination (ΔG = -40.8 kcal/mol) is thermodynamically favored over heme-O₂ coordination (ΔG = -23.3 kcal/mol) (Fig. 7d). Consistently, activity assays with CO-pretreated heme solutions showed minimal enzyme catalysis in Rn HO-1 Δ22 (Fig. 7b). These results, in combination with QM calculations indicating strong coordination between heme and both O₂ and CO ligands, suggest that free heme is unlikely to persist in aerobic solvent environments. To further explore the effects of CO on enzyme activity, we performed molecular dynamics simulations. These simulations revealed distinct binding free energies of -8.53 kcal/mol for heme-O₂ and -4.51 kcal/mol for heme-CO (Fig. 7e), suggesting that the enzyme has a significantly weaker affinity for heme-CO. Per-residue decomposition analysis of the binding free energies identified key residues around the substrate-binding pocket that contribute to this difference (Fig. 7f and 7g). Notably, Ser142, which is located near the CO ligand, exhibited higher energy levels in the heme-CO complex compared to heme-O₂, suggesting substantial steric hindrance. This hindrance arises from the rigid, linear geometry of the sp-hybridized CO ligand, in contrast to the naturally bent sp²-hybridized O₂ ligand, which adopts an optimal bond angle to minimize spatial conflicts. Energy differences between heme-CO and heme-O₂ binding configurations across all residues are shown in Fig. 7h. The free energy landscape constructed from the RMSD and radius of gyration (Supplementary Fig. 19) during the stable phase of the simulation revealed a deeper and broader energy well for the heme-O₂-enzyme complex (Fig. 7i) compared to the heme-CO complex (Fig. 7j), further supporting the thermodynamic preference for heme-O₂. Our computational analyses provide a mechanistic explanation for CO-induced inhibition of heme utilization in Rn HO-1 Δ22 : the formation of stable heme-CO complexes impedes proper substrate positioning within the catalytic pocket, blocking the subsequent O₂-dependent activation of the reaction intermediate. This competitive coordination mechanism is likely exacerbated by CO's accumulation in the solvent environment, where it functions as a potent heme-sequestering agent, outcompeting O₂ for coordination and hindering enzyme catalysis. Efficient bilirubin synthesis enabled by CO removal and NADPH regeneration To mitigate CO-mediated inhibition, we introduced an engineered carbon monoxide dehydrogenase ( Ch CODH) 52 to oxidize CO into non-toxic CO₂. Additionally, to enable cofactor regeneration, we incorporated an engineered NADPH-dependent formate dehydrogenase ( Ap FDH) 53 . For multi-enzyme co-expression, we used pCDFDuet-1 and pETDuet-1 plasmids, adding an extra ribosome binding site (RBS) on pETDuet-1. This strategy led to the construction of a five-enzyme co-expression strain that co-expressed Rn HO-1 Δ22 , Rn CPR Δ54 , Rn BVR, Ap FDH, and Ch CODH (Supplementary Fig. 20). A schematic of the multi-enzyme cascade catalysis system is shown in Fig. 8b. This optimized cascade system successfully converted heme to bilirubin (BR), achieving complete conversion of 1953 mg/L (3 mM) heme to 1678.4 mg/L (2.87 mM) BR, resulting in a 95.8% yield in a 1 L reaction system (Fig. 8d). A color transition from dark brown to reddish-brown was observed throughout the process (Fig. 8a, 8c). BR was extracted from the reaction mixture via chloroform extraction, followed by rotary evaporation and lyophilization. The resulting powder was analyzed by NMR spectroscopy, with the 1 H and 13 C NMR spectra confirming that the synthesized BR adopts the IXα configuration (Supplementary Fig. 21 and 22). In contrast to previous reports, this study represents the first successful enhancement of biosynthesized bilirubin production to gram-scale quantities (Fig. 8e). While existing research on bilirubin biosynthesis often utilizes readily available carbon sources such as glucose, which offers cost-effectiveness, our optimized system achieved a remarkable 20-fold higher yield compared to previous studies. Furthermore, the rational truncation strategy employed for improved protein expression efficiency, alongside the systematic byproduct elimination protocol developed here, provides valuable insights for metabolic engineering strategies aimed at optimizing bilirubin biosynthesis. Discussion This study redefines the enzymatic synthesis of BR by addressing two long-standing yet previously uncharacterized bottlenecks—Fe²⁺-mediated oxidative degradation and CO-induced enzyme inhibition—while achieving record-breaking production titers. The insights and strategies developed herein not only overcome critical limitations in BR biosynthesis but also provide a broadly applicable blueprint for optimizing complex enzymatic cascades in synthetic biology and industrial biocatalysis. A central contribution lies in the mechanistic elucidation of Fe²⁺-induced product instability, a phenomenon not previously recognized in BR synthesis systems. Earlier studies attributed low yields to enzymatic inefficiency or substrate inhibition, overlooking the role of byproduct-metal interactions. Through DFT and TD-DFT analyses, we demonstrated that Fe²⁺ coordinates with deprotonated biliverdin (deBV) and bilirubin (deBR), forming reactive intermediates prone to radical-driven oxidative degradation (Fig. 5). This finding challenges the conventional view of Fe²⁺ as a benign byproduct and establishes metal chelation as an essential strategy for stabilizing tetrapyrrole biosynthesis. The combined use of etidronic acid (HEDP) and pH modulation to suppress deprotonation offers a novel, universally applicable approach to mitigating metal-mediated degradation in porphyrin-based pathways—a strategy previously absent for BR production efforts. Equally significant is the identification of CO as a catalytic poison in heme oxygenase (HO)-mediated systems. While CO’s affinity for heme is well-documented in respiratory biology, its inhibitory effect on HO catalysis had not been mechanistically dissected. Our molecular dynamics simulations revealed that CO’s linear geometry induces steric clashes with key residues (e.g., Ser142), thereby hindering substrate access to the HO active site. This discovery has broad implications for heme-dependent enzymes beyond BR synthesis. The incorporation of Ch CODH to actively eliminate CO represents the first successful application of gas-phase byproduct removal in BR biosynthesis and offers a scalable solution to a long-standing but overlooked issue in gas-ligand and heme-containing enzymatic systems. The yield achieved in this study—1,678 mg/L BR at 95.8% yield—marks a paradigm shift in enzymatic BR production. Comparation with previous reports underscores this advance: earlier enzymatic systems peaked at 325 mg/L 34 , while de novo microbial approaches struggled to exceed 75.5 mg/L 30 . This 20-fold enhancement stems from addressing dual byproduct interferences—a factor neglected in prior efforts that focused on enzyme overexpression or cofactor supplementation 29,31,33 . For instance, while Liu et al. 29 attributed yield limitations to HO-1 kinetics, our work demonstrates that even optimized oxygenase activity cannot overcome unchecked Fe²⁺- and CO-mediated losses. These comparisons highlight the necessity of holistic pathway optimization that considers both enzymatic and non-enzymatic factors. Beyond BR synthesis, this study offers methodological innovations with cross-disciplinary relevance. The integration of DFT-guided mechanistic prediction and enzymatic byproduct management exemplifies a "design-test-learn" strategy applicable to other metalloenzyme vulnerable to metal toxicity or gas-phase inhibition. For example, cytochrome P450 cascades, which also generate CO and reactive metals 55 , could benefit from Ch CODH or targeted chelators to enhance stability and performance. Furthermore, the rational truncation of membrane-associated domains in Rn HO-1 and Rn CPR provides a practical strategy for solubilizing structurally challenging oxidoreductases, a persistent bottleneck in industrial enzymology 56 . In conclusion, this work transcends incremental advances by unraveling and resolving two previously "invisible" mechanisms constrained BR biosynthesis. By shifting the focus from isolated enzyme engineering to systemic byproduct management, we establish a new paradigm for pathway optimization—one that improves catalytic efficiency with chemical robustness. These findings not only enable gram-scale BR production but also redefine the boundaries of synthetic biology, proving that computational mechanistic insights and enzymatic innovation can synergistically unlock industrial-scale synthesis of high-value natural products. Methods Materials All wild-type gene sequences were codon-optimized, synthesized, and cloned into the Bam H I/ Xho I restriction sites of the pET28a(+) vector by Sangon Biotech (Shanghai, China). PrimeSTAR Max DNA Polymerase and the restriction enzyme Dpn I were obtained from Takara Bio Inc. (Japan). The homologous recombination and DNA purification kits were purchased from Vazyme Biotech Co., Ltd. (China). The SDS-PAGE gel preparation kit was sourced from Sangon Biotech (Shanghai, China). NADPH was acquired from Roche Holding AG (Switzerland), and heme (hemin chloride, molecular weight = 651.94 Da) was purchased from Aladdin Industrial Corporation (China). Plasmids pETDuet-1, pCDFDuet-1, and E. coli BL21 (DE3) were preserved in our laboratory stock. All other reagents were sourced from Shanghai Macklin Biochemical Co., Ltd. (China), unless otherwise specified. Phylogenetic analysis Phylogenetic trees were constructed using the Neighbor–Joining method in Molecular Evolutionary Genetics Analysis version 11 (MEGA11) 57 . Protein sequences used for tree construction were retrieved from the NCBI database, with selected sequences (Supplementary Table 1) representing diverse biological origins and catalytic properties. The tree was visualized using the Chiplot online platform 58 . Protein expression and purification Recombinant plasmids were transformed into E. coli BL21(DE3) strains for protein expression. E. coli cultures were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) with kanamycin or ampicillin (50 μg/mL) at 37°C with shaking (200 rpm). Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM when the OD 600 reached 0.4‒0.6. After 14 hours of induction at 20°C, cells were collected by centrifugation (8,000 × g ), and SDS-PAGE analysis was performed to verify protein expression levels. For enzyme purification, cells were resuspended in 100 mM Tris-HCl buffer (pH 7.5) and disrupted by ultrasonication on ice. The supernatant was obtained by centrifugation (12,000 × g for 10 min at 4°C) and purified using Ni²⁺ affinity chromatography, as previously described 59 . Enzyme screening Reactions for HO and its partner proteins CPR or FDR were conducted by adding 100 mg/L HO and either 100 mg/L CPR or 100 mg/L FDR, with 1 mM heme and 10 mM NADPH, followed by incubation at 35°C with agitation (600 rpm) for 1 hour. Reactions were quenched by a 10-fold dilution with DMSO, and product concentrations were analyzed by HPLC. For BVR screening, reactions containing 100 mg/L BVR, 1 mM BV, and 2 mM NADPH were conducted at 35°C with agitation (600 rpm) for 30 minutes, followed by quenching with DMSO dilution, and product levels were determined by HPLC. Generation of truncated mutants Truncated plasmids were generated by amplifying gene sequences with the primer pairs listed in Supplementary Table 2, using pET28a(+)- Rn HO-1 or pET28a(+)- Rn CPR as templates, following a previously reported PCR protocol 60 . The PCR products were treated with Dpn I to remove template plasmids, purified, and subjected to homologous recombination using a Vazyme Biotech kit. The resulting plasmids were transformed into E. coli BL21 (DE3), and colonies were screened for the correct mutants, which were validated by DNA sequencing (Sangon Biotech, Shanghai, China). LC-MS analysis LC-MS analysis of BV and BR degradation products was performed using a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer. Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm particle size; Waters Corporation, Milford, MA, USA) with electrospray ionization (ESI) in positive ion mode. The gradient elution program was as follows: 0‒5 min: 20% B; 5‒10 min: 20%‒40% B; 10‒15 min: 40% B; 15‒20 min: 40%‒100% B; 20‒30 min: 100% B; 30‒32.5 min: 100%‒20% B; 32.5‒35 min: 20% B. The column temperature was maintained at 30°C, and the flow rate was 0.2 mL/min. Data were processed using Xcalibur software (Thermo Fisher Scientific). Screening of chelators For initial chelator screening, 1 mM BV or BR standard solutions were mixed with 5 mM chelator candidates (tartrate, citrate, EDTA, DEG, HEDP, NTA, HEDTA) and 1 mM FeCl₂. After incubation at 35°C for 150 minutes, samples were analyzed by UV-Vis spectroscopy (Supplementary Methods). To evaluate chelator effects on enzymatic activity, crude enzyme reactions were conducted with Rn HO-1 Δ22 , Rn CPR Δ54 , or Rn HO-1 Δ22 , Rn CPR Δ54 , and Rn BVR in the presence of 5 mM of each chelator. Reactions were performed under conditions (1 mM heme, 10 mM NADPH, 100 mM Tris-HCl, pH 7.5, 35°C, 600 rpm), and products were quantified by HPLC. Theoretical calculations Density functional theory (DFT) calculations were performed using Gaussian 09, Revision D.01 61 . Initial molecular structures were modeled in GaussView 5.0 and subjected to conformational searches with the Molclus program 62 . Geometry optimizations and vibrational frequency analyses were performed at the B3LYP/6-311G(d,p) level of theory, and single-point energy calculations were carried out at the BP86/def2-TZVP level. A polarizable continuum model (PCM) was used to simulate aqueous solvation effects, and Grimme’s GD3BJ dispersion correction was applied. For Fe²⁺-containing systems, all possible spin multiplicities were evaluated to determine the ground-state configuration. UV-Vis absorption spectra for BV were calculated using time-dependent DFT (TD-DFT) at the BP86/def2-TZVP level, with 50 excited states computed. Electrostatic potential (ESP) charges and frontier molecular orbitals (FMOs) were analyzed using the Multiwfn program (Version 3.8) 63 , and results were visualized using VMD software (Version 1.9.3) 64 . Scaled-up BR synthesis For scaled-up BR synthesis, the reaction was conducted in a 5 L bioreactor (T&J Bioengineering, Shanghai, China). The system contained 20 g dry cell weight (DCW)/L of lysate from the five-enzyme co-expression strain E. coli /pCDFDuet-1- Rn CPR Δ54 / Rn HO-1 Δ22 /pETDuet-1- Ch CODH/ Rn BVR/ Ap FDH, 1953 mg/L (3 mM) heme, 50 mM ammonium formate, 0.1 mM FMN, and 15 mM HEDP. The reaction was conducted at 35°C with agitation (400 rpm), with pH automatically maintained at 6.5 ± 0.1 by controlled addition of HCl, and air was continuously supplied at 2 vvm (volume per volume per minute). Statistical analysis Each experiment was performed at least in triplicates. Values were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using Origin2024 with student’s t-test. And a value of p < 0.05 was considered statistically significant. Declarations Data availability All data is included in the main text or Supplementary Information file. Source data are provided with this paper. Acknowledgements The authors greatly appreciate the financial support from the National Key Research and Development Program of China (2023YFA0914500), the National Science Foundation of China (32271487), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE201812), and the Program of Introducing Talents of Discipline to Universities (111-2-06). Author contributions Z.T.J. and J.X.R. contributed equally to this work. Z.T.J. designed the experiments, analyzed the data, conducted DFT calculation and wrote the first draft. J.X.R. contributed to molecular dynamics simulation, analysis of simulation results and energy calculation. C.K.Z contributed to cell fermentation and protein purification. Y.M.L contributed to generation of mutants. Q.Z contributed to the process of scale-up reaction. W.C.Z. and M.Y.Z. reviewed the manuscript and gave the proferssional suggestions. R.Z.Z. guided the project and revised the manuscript. All authors read and approved the manuscript. References Tell, G. & Gustincich, S. Redox state, oxidative stress, and molecular mechanisms of protective and toxic effects of bilirubin on cells. Curr. Pharm. Design 15 , 2908-2914 (2009). https://doi.org:10.2174/138161209789058174 Du, L. et al. Neonatal hyperbilirubinemia management: Clinical assessment of bilirubin production. Semin. Perinatol. 45 , 151351 (2021). https://doi.org:10.1016/j.semperi.2020.151351 Hinds, T. D., Jr. & Stec, D. E. Bilirubin safeguards cardiorenal and metabolic diseases: a protective role in health. Curr. Hypertens. Rep. 21 , 87 (2019). https://doi.org:10.1007/s11906-019-0994-z Ziberna, L., Martelanc, M., Franko, M. & Passamonti, S. Bilirubin is an endogenous antioxidant in human vascular endothelial cells. Sci. Rep. 6 , 29240 (2016). https://doi.org:10.1038/srep29240 Jayanti, S., Vitek, L., Tiribelli, C. & Gazzin, S. The Role of bilirubin and the other "yellow players" in neurodegenerative diseases. Antioxidants 9 , 900 (2020). https://doi.org:10.3390/antiox9090900 Khurana, I. et al. Can bilirubin nanomedicine become a hope for the management of COVID-19? Med. Hypotheses 149 , 110534 (2021). https://doi.org:10.1016/j.mehy.2021.110534 Yu, Z. J. et al. Calculus bovis: A review of the traditional usages, origin, chemistry, pharmacological activities and toxicology. J. Ethnopharmacol. 254 , 112649 (2020). https://doi.org:10.1016/j.jep.2020.112649 Wang, D. Q. H. & Carey, M. C. Therapeutic uses of animal biles in traditional Chinese medicine: An ethnopharmacological, biophysical chemical and medicinal review. World J. Gastroenterol. 20 , 9952-9975 (2014). https://doi.org:10.3748/wjg.v20.i29.9952 Zhang, J. et al. Recent advances in microbial production of high-value compounds in the tetrapyrrole biosynthesis pathway. Biotechnol. Adv. 55 , 107904 (2022). https://doi.org:10.1016/j.biotechadv.2021.107904 Ko, Y. J. et al. Bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories: engineering, metabolic regulations, challenges, and perspectives. Crit. Rev. Biotechnol. , 44 , 373-387 (2023). https://doi.org:10.1080/07388551.2023.2168512 Waza, A. A., Hamid, Z., Ali, S., Bhat, S. A. & Bhat, M. A. A review on heme oxygenase-1 induction: is it a necessary evil. Inflamm. Res. 67 , 579-588 (2018). https://doi.org:10.1007/s00011-018-1151-x Pena, A. C. & Pamplona, A. Heme oxygenase-1, carbon monoxide, and malaria-The interplay of chemistry and biology. Coord. Chem. Rev. 453 , 214285 (2022). https://doi.org:10.1016/j.ccr.2021.214285 Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 114 , 3919-3962 (2014). https://doi.org:10.1021/cr400415k Salim, M., Brown-Kipphut, B. A. & Maines, M. D. Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation. J. Biol. Chem. 276 , 10929-10934 (2001). https://doi.org:10.1074/jbc.M010753200 Florczyk, U. M., Jozkowicz, A. & Dulak, J. Biliverdin reductase: new features of an old enzyme and its potential therapeutic significance. Pharmacol. Rep. 60 , 38-48 (2008). Matsui, T., Iwasaki, M., Sugiyama, R., Unno, M. & Ikeda-Saito, M. Dioxygen activation for the self-degradation of heme: reaction mechanism and regulation of heme oxygenase. Inorg. Chem. 49 , 3602-3609 (2010). https://doi.org:10.1021/ic901869t Sato, H. et al. Crystal structure of rat haem oxygenase-1 in complex with ferrous verdohaem: presence of a hydrogen-bond network on the distal side. Biochem. J. 419 , 339-345 (2009). https://doi.org:10.1042/BJ20082279 Hamdane, D. et al. Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450. J. Biol. Chem. 284 , 11374-11384 (2009). https://doi.org:10.1074/jbc.M807868200 Sugishima, M., Wada, K. & Fukuyama, K. Recent advances in the understanding of the reaction chemistries of the heme catabolizing enzymes HO and BVR Based on high resolution protein structures. Curr. Med. Chem. 27 , 3499-3518 (2020). https://doi.org:10.2174/0929867326666181217142715 Tohda, R. et al. Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin. J. Biol. Chem. 296 , 100217 (2021). https://doi.org:10.1074/jbc.RA120.016271 Wang, J. et al. Enzymological and structural characterization of Arabidopsis thaliana heme oxygenase-1. FEBS Open Bio 12 , 1677-1687 (2022). https://doi.org:10.1002/2211-5463.13453 Castrignano, S. et al. Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops. Biochim. Biophys. Acta Proteins Proteom. 1866 , 116-125 (2018). https://doi.org:10.1016/j.bbapap.2017.07.009 Paukovich, N. et al. Biliverdin reductase B dynamics are coupled to coenzyme binding. J. Mol. Biol. 430 , 3234-3250 (2018). https://doi.org:10.1016/j.jmb.2018.06.015 Takao, H. et al. A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity. Nat. Commun. 8 , 14397 (2017). https://doi.org:10.1038/ncomms14397 Ishchuk, O. P. et al. Genome-scale modeling drives 70-fold improvement of intracellular heme production in Saccharomyces cerevisiae . Proc. Natl. Acad. Sci. USA 119 , e2108245119 (2022). https://doi.org:10.1073/pnas.2108245119 Choi, K. R., Yu, H. E., Lee, H. & Lee, S. Y. Improved production of heme using metabolically engineered Escherichia coli . Biotechnol. Bioeng. 119 , 3178-3193 (2022). https://doi.org:10.1002/bit.28194 Chen, H. et al. High-yield porphyrin production through metabolic engineering and biocatalysis. Nat. Biotechnol. (2024). https://doi.org:10.1038/s41587-024-02267-3 Yang, S. et al. Recent advances in microbial synthesis of free heme. Appl. Microbiol. Biotechnol. 108 , 17-27 (2024). https://doi.org:10.1007/s00253-023-12968-5 Liu, Z., Xiong, P., Guo, N. & Chen, H. Efficient biosynthesis of bilirubin by overexpressing heme oxygenase, biliverdin reductase and 5-aminolevulinic acid dehydratase in Escherichia coli . Mol. Catal. 571 , 114714 (2025). https://doi.org:10.1016/j.mcat.2024.114714 Chen, H., Xiong, P., Guo, N. & Liu, Z. Metabolic engineering of Escherichia coli for production of a bioactive metabolite of bilirubin. Int. J. Mol. Sci. 25 (2024). https://doi.org:10.3390/ijms25179741 Mei, J. et al. Production of biliverdin by biotransformation of exogenous heme using recombinant Pichia pastoris cells. Bioresour. Bioprocess. 11 , 19 (2024). https://doi.org:10.1186/s40643-024-00736-w Yan, S. et al. Efficient production of biliverdin through whole-cell biocatalysis using recombinant Escherichia coli . Chinese J. biotechnol. 38 , 2581-2593 (2022). https://doi.org:10.13345/j.cjb.220137 Chen, D., Brown, J. D., Kawasaki, Y., Bommer, J. & Takemoto, J. Y. Scalable production of biliverdin IX alpha by Escherichia coli . BMC Biotechnol. 12 , 89 (2012). https://doi.org:10.1186/1472-6750-12-89 Mei, J. et al. Production of bilirubin by biotransformation of biliverdin using recombinant Escherichia coli cells. Bioprocess. Biosyst. Eng. 45 , 563-571 (2022). https://doi.org:10.1007/s00449-021-02679-4 Kepp, K. P. Heme: From quantum spin crossover to oxygen manager of life. Coord. Chem. Rev. 344 , 363-374 (2017). https://doi.org:10.1016/j.ccr.2016.08.008 Szterenberg, L., Latos‐Grażyński, L. & Wojaczyński, J. Metallobiliverdin radicals—DFT studies. ChemPhysChem 4 , 691-698 (2003). https://doi.org:10.1002/cphc.200200611 Dimitrijević, M. S. et al. Biliverdin–copper complex at physiological pH. Dalton Trans. 48 , 6061-6070 (2019). https://doi.org:10.1039/c8dt04724c Martínez, A., López-Rull, I. & Fargallo, J. A. To prevent oxidative stress, what about protoporphyrin IX, biliverdin, and bilirubin? Antioxidants 12 , 1662 (2023). https://doi.org:10.3390/antiox12091662 Bozic, B. et al. Mechanisms of redox interactions of bilirubin with copper and the effects of penicillamine. Chem. Biol. Interact. 278 , 129-134 (2017). https://doi.org:10.1016/j.cbi.2017.10.022 Liu, S., Xia, S., Yue, D., Sun, H. & Hirao, H. The bonding nature of Fe–CO complexes in heme proteins. Inorg. Chem. 61 , 17494-17504 (2022). https://doi.org:10.1021/acs.inorgchem.2c02387 Medina, M. V., Sapochnik, D., Garcia Sola, M. & Coso, O. Regulation of the expression of heme oxygenase-1: Signal transduction, gene promoter activation, and beyond. Antioxid. Redox Signal. 32 , 1033-1044 (2020). https://doi.org:10.1089/ars.2019.7991 Uchida, T., Sekine, Y., Matsui, T., Ikeda-Saito, M. & Ishimori, K. A heme degradation enzyme, HutZ, from Vibrio cholerae . Chem. Commun. 48 , 6741-6743 (2012). https://doi.org:10.1039/c2cc31147j Sugishima, M. et al. Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase. Proc. Natl. Acad. Sci. USA 111 , 2524-2529 (2014). https://doi.org:10.1073/pnas.1322034111 Duff, M. R. et al. Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family. J. Biochem. 168 , 191-202 (2020). https://doi.org:10.1093/jb/mvaa039 O'Brien, L., Hosick, P. A., John, K., Stec, D. E. & Hinds, T. D., Jr. Biliverdin reductase isozymes in metabolism. Trends Endocrinol. Metab. 26 , 212-220 (2015). https://doi.org:10.1016/j.tem.2015.02.001 Ahmed, F. H. et al. Rv2074 is a novel F 420 H 2 -dependent biliverdin reductase in Mycobacterium tuberculosis . Protein Sci. 25 , 1692-1709 (2016). https://doi.org:10.1002/pro.2975 Ryter, S. W. Heme oxygenase-1: An anti-inflammatory effector in cardiovascular, lung, and related metabolic disorders. Antioxidants 11 , 555 (2022). https://doi.org:10.3390/antiox11030555 Stanojevic, J. S., Zvezdanovic, J. B. & Markovic, D. Z. Bilirubin degradation in methanol induced by continuous UV-B irradiation: a UHPLC-ESI-MS study. Pharmazie 70 , 225-230 (2015). https://doi.org:10.1691/ph.2015.4122 Ritter, M. et al. Isolation and identification of intermediates of the oxidative bilirubin degradation. Org. Lett. 18 , 4432-4435 (2016). https://doi.org:10.1021/acs.orglett.6b02287 Chung, L. W., Li, X., Sugimoto, H., Shiro, Y. & Morokuma, K. Density functional theory study on a missing piece in understanding of heme chemistry: The reaction mechanism for indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. J. American Chem. Soc. 130 , 12299-12309 (2008). https://doi.org:10.1021/ja803107w Zazzeron, L., Franco, W. & Anderson, R. Carbon monoxide poisoning and phototherapy. Nitric Oxide-Biol. Chem. 146 , 31-36 (2024). https://doi.org:10.1016/j.niox.2024.04.001 Kim, S. M. et al. O2-tolerant CO dehydrogenase via tunnel redesign for the removal of CO from industrial flue gas. Nat. Catal. 5 , 807-817 (2022). https://doi.org:10.1038/s41929-022-00834-y Cheng, F. et al. Switching the cofactor preference of formate dehydrogenase to develop an NADPH-dependent biocatalytic system for synthesizing chiral amino acids. J. Agric. Food Chem. 71 , 9009-9019 (2023). https://doi.org:10.1021/acs.jafc.3c01561 Zhou, W. et al . Production of bilirubin via whole-cell transformation utilizing recombinant Corynebacterium glutamicum expressing a beta-glucuronidase from S taphylococcus sp. RLH1. Biotechnol. Lett. 46 , 223-233 (2024). https://doi.org:10.1007/s10529-024-03468-1 Yuan, Z. N., Cruz, L. K. D., Yang, X. X. & Wang, B. H. Carbon monoxide signaling: examining its engagement with various molecular targets in the context of binding affinity, concentration, and biologic responses. Pharmacol. Rev. 74 , 823-873 (2022). https://doi.org:10.1124/pharmrev.121.000564 Kaur, J., Kumar, A. & Kaur, J. Strategies for optimization of heterologous protein expression in E. coli : Roadblocks and reinforcements. Int. J. Biol. Macromol. 106 , 803-822 (2018). https://doi.org:10.1016/j.ijbiomac.2017.08.080 Tamura, K., Stecher, G. & Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 38 , 3022-3027 (2021). https://doi.org:10.1093/molbev/msab120 Xie, J. et al. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res. 51 , W587-W592 (2023). https://doi.org:10.1093/nar/gkad359 Ruan, Y. Q., Zhang, R. Z. & Xu, Y. Directed evolution of maltogenic amylase from Bacillus licheniformis R-53: Enhancing activity and thermostability improves bread quality and extends shelf life. Food Chem. 381 , 132222 (2022). https://doi.org:10.1016/j.foodchem.2022.132222 Xi, Z. W. et al. Deciphering the key loop: enhancing l-threonine transaldolase's catalytic potential. Acs Catal. 14 , 10462-10474 (2024). https://doi.org:10.1021/acscatal.4c02049 Gaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2013. Lu T, Molclus program, version 1.12, http://www.keinsci.com/research/molclus.html Lu, T. & Chen, F. Multiwfn: a multifunctional wavefunction analyzer. J. Comput. Chem. 33 , 580-592 (2012). https://doi.org:10.1002/jcc.22885 Humphrey, W., Dalke, A. & Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graph. 14 , 33-38 (1996). https://doi.org:10.1016/0263-7855(96)00018-5 Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx Supplementary Information Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6554107","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":452508573,"identity":"f67452a5-9515-4f25-b072-a04b22e553f1","order_by":0,"name":"Rongzhen Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYHACNhAhx9iMEDEgSosx6VoSG5BE8Gsx7z/+7MHPHbXpze3Mzx5+3WGXx8DevE2CoeYOTi0yBw6kG/aeOZ7b2Mxmbix7JrmYgedYmQTDsWc4tUgwNhyT4G07BtTCYCYt2cac2CCRYwYUPYxbCzNjm+TftmPpjM3s34Ba6hMb5N8Q0MLGzCbN21aTwNjMYyb5se0w0BYeAlp42NikZdsOGDY285RJM7YdT2zjSSu2SDiGRwv/8WeSb9vq5A37j2+T/NlWndjPfnjjjQ81uLVAwWEGwwYGBmYeBmg0JRDSwMBQxyAPJBl/EFY5CkbBKBgFIxAAAOKzTsod7FCZAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5745-1190","institution":"Jiangnan university","correspondingAuthor":true,"prefix":"","firstName":"Rongzhen","middleName":"","lastName":"Zhang","suffix":""},{"id":452508574,"identity":"796d095a-40ef-4905-8704-ee72bb963802","order_by":1,"name":"Zhentao Jiang","email":"","orcid":"","institution":"Jiangnan university","correspondingAuthor":false,"prefix":"","firstName":"Zhentao","middleName":"","lastName":"Jiang","suffix":""},{"id":452508575,"identity":"8e02388a-5ce9-4cb7-957a-c942e8dd0eef","order_by":2,"name":"Jingxin Rao","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Jingxin","middleName":"","lastName":"Rao","suffix":""},{"id":452508576,"identity":"335dd0a2-424c-4678-9b7a-cb2a86a9a65d","order_by":3,"name":"Caokai Zhu","email":"","orcid":"","institution":"Jiangnan university","correspondingAuthor":false,"prefix":"","firstName":"Caokai","middleName":"","lastName":"Zhu","suffix":""},{"id":452508577,"identity":"3b4b306e-d108-4fc9-942c-b8b9f63837f9","order_by":4,"name":"Yamiao Li","email":"","orcid":"","institution":"Jiangnan university","correspondingAuthor":false,"prefix":"","firstName":"Yamiao","middleName":"","lastName":"Li","suffix":""},{"id":452508578,"identity":"5f17b08a-85fc-480e-bee5-eedab7cd81b2","order_by":5,"name":"Qiang Zhu","email":"","orcid":"","institution":"Jiangnan university","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Zhu","suffix":""},{"id":452508579,"identity":"3e98a46d-4f03-4732-93a2-5dfc7c51b4db","order_by":6,"name":"Mingyue Zheng","email":"","orcid":"https://orcid.org/0000-0002-3323-3092","institution":"Shanghai Institute of Materia Medica, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Zheng","suffix":""},{"id":452508580,"identity":"feb3e9ca-2b9e-4ca5-a585-5861adf465fb","order_by":7,"name":"Wenchi Zhang","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Wenchi","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-04-29 08:21:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6554107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6554107/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67804-3","type":"published","date":"2025-12-23T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83612154,"identity":"b793f5d0-99ee-4535-b29a-1b28a57bd13d","added_by":"auto","created_at":"2025-05-29 12:36:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":145602,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnzymatic conversion of heme to bilirubin and structural features of key pathway enzymes.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003ea\u003c/strong\u003e, Overall reaction pathway from heme to bilirubin (BR), catalyzed sequentially by heme oxygenase (HO) and biliverdin reductase (BVR).\u003cbr\u003e\n \u003cstrong\u003eb\u003c/strong\u003e, Conformational dynamics of HO and its redox partner (RP) during electron transfer: the closed conformation enables electron transfer from NADPH to FMN, while the open conformation facilitates FMN-mediated electron delivery to the heme-bound HO.\u003cbr\u003e\n \u003cstrong\u003ec\u003c/strong\u003e, Mechanistic steps of HO-catalyzed heme degradation, including O₂ activation and porphyrin ring cleavage, yielding biliverdin (BV), Fe²⁺, and carbon monoxide (CO).\u003cbr\u003e\n \u003cstrong\u003ed\u003c/strong\u003e, Alternative forms of redox partners across species, including separate FAD- and FMN-containing proteins that mediate electron transfer \u003cem\u003evia\u003c/em\u003eFe–S clusters.\u003cbr\u003e\n \u003cstrong\u003ee\u003c/strong\u003e, Structural architecture of BVR, highlighting the Rossmann-fold domain and binding pockets for NADPH and biliverdin.\u003cbr\u003e\n \u003cstrong\u003ef\u003c/strong\u003e, Proposed mechanism for BV reduction by BVR, involving π-stacked BV molecules and NADPH-mediated hydride transfer.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/40f48d055067f19cb49fec71.jpg"},{"id":83612721,"identity":"6bcaa652-68ab-497d-9cdf-291004e73119","added_by":"auto","created_at":"2025-05-29 12:44:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":187859,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of key enzymes for bilirubin biosynthesis.\u003c/strong\u003e\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003ePhylogenetic tree of heme oxygenases (HOs) constructed to capture evolutionary diversity.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e Phylogenetic analysis of redox partners (RPs), including both single- and two-component systems.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e Phylogenetic analysis of biliverdin reductases (BVRs), encompassing both animal and bacterial isoforms. Enzymes selected for experimental screening are marked with red stars.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003eActivity screening of various HO-RP combinations for heme-to-biliverdin conversion; the combination of \u003cem\u003eRattus norvegicus\u003c/em\u003e HO-1 and CPR (\u003cem\u003eRn\u003c/em\u003eHO-1/\u003cem\u003eRn\u003c/em\u003eCPR) showed the highest BV yield.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ee\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003eScreening of BVRs for biliverdin-to-bilirubin conversion, with \u003cem\u003eRn\u003c/em\u003eBVR exhibiting the highest catalytic efficiency.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/7b338e7f8a84572fca7c137f.jpg"},{"id":83612722,"identity":"ca6383f0-462d-4c62-aadd-390f9e0d946f","added_by":"auto","created_at":"2025-05-29 12:44:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTerminal truncation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eHO-1 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eCPR improves soluble expression without compromising enzymatic activity.\u003c/strong\u003e\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eHydrophobicity analysis of \u003cem\u003eRn\u003c/em\u003eCPR and RnHO-1 reveals hydrophobic membrane-associating regions at the N-terminus of \u003cem\u003eRn\u003c/em\u003eCPR (residues 1–54) and C-terminus of \u003cem\u003eRn\u003c/em\u003eHO-1 (residues 268–289). These regions were systematically truncated to enhance soluble expression.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eb,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eSDS-PAGE analysis of soluble expression levels in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) shows increased expression with progressive truncation, with \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003eΔ54\u003c/sub\u003e and \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e exhibiting the highest solubility.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ec,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eMichaelis–Menten kinetic parameters (\u003cem\u003eK\u003c/em\u003eₘ and \u003cem\u003ek\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003eₐₜ/\u003cem\u003eK\u003c/em\u003eₘ) of wild-type and truncated \u003cem\u003eRn\u003c/em\u003eHO-1 and \u003cem\u003eRn\u003c/em\u003eCPR variants. All truncation mutants retain comparable catalytic efficiency. \u003cem\u003eRn\u003c/em\u003eBVR is included as a control; kinetic parameters were not determined (N.D.) due to strong substrate inhibition. N.A., not applicable.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/99c08c9d1eb0f559ed104ffd.jpg"},{"id":83612159,"identity":"dbfef537-adea-4287-a462-42a0cb6cd803","added_by":"auto","created_at":"2025-05-29 12:36:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnzymatic conversion through stepwise and one-pot cascade reactions.\u003c/strong\u003e\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eTime-course analysis of biliverdin (BV) production from heme using wild-type enzymes and optimized truncation variants (\u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e/\u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003eΔ54\u003c/sub\u003e), demonstrating enhanced catalytic performance of the engineered pair.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eb,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eTime-dependent conversion of BV to bilirubin (BR) catalyzed by \u003cem\u003eRn\u003c/em\u003eBVR, confirming efficient reductase activity.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ec,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eOne-pot enzymatic cascade converting heme to BR using the optimized \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e/\u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003eΔ54\u003c/sub\u003e pair and \u003cem\u003eRn\u003c/em\u003eBVR, illustrating seamless integration of the two-step pathway for bilirubin biosynthesis.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/f87f77784c0fb053047831b8.jpg"},{"id":83612161,"identity":"ef0b5c3c-8730-4cdc-8b2a-2d4c09367324","added_by":"auto","created_at":"2025-05-29 12:36:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Fe²⁺ on the stability of biliverdin (BV) and bilirubin (BR).\u003c/strong\u003e\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eHPLC chromatograms showing the degradation of BV upon incubation with Fe²⁺, indicating stability loss.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eb,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eHPLC chromatograms of BR incubated with Fe²⁺, demonstrating similar instability and degradation.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ec,\u003c/strong\u003e\u003c/em\u003e Density functional theory (DFT)-calculated free energies of Fe²⁺ coordination with deprotonated BV (deBV) and BR (deBR), illustrating the strength of coordination.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ed,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eFree energy of axial ligand (O₂/OH⁻) chelation to Fe²⁺ in the deBV and deBR complexes, revealing that axial ligands are prone to chelation with Fe\u003csup\u003e2+\u003c/sup\u003e.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ee,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eFrontier molecular orbitals (HOMO/LUMO) of deBV, deBV-Fe²⁺, and the case with axial ligands (isovalue = 0.02), highlighting the electronic characteristics on different atoms.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ef,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eFrontier molecular orbitals (HOMO/LUMO) of deBR-Fe²⁺ with axial ligands (isovalue = 0.02), showing similar electronic characteristics to the series of deBV complexes.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eg,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eProposed mechanism of Fe²⁺-induced oxidative ring-opening degradation of BV and BR, outlining the critical steps in degradation triggered by Fe²⁺ coordination.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/23f1dbef8b343d23cf3e6bd9.jpg"},{"id":83612155,"identity":"12561c49-8531-4829-9d7b-1039deb2833c","added_by":"auto","created_at":"2025-05-29 12:36:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":96003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of chelating agents and protonation state adjustment to prevent product degradation.\u003c/strong\u003e\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eEffect of various chelating agents on HO-catalyzed heme-to-biliverdin (BV) conversion, with statistical significance indicated by ***p \u0026lt; 0.001.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003eb,\u003c/strong\u003e\u003c/em\u003e Effect of chelating agents on the HO/BVR cascade-catalyzed heme-to-bilirubin (BR) conversion, ***p \u0026lt; 0.001.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ec,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eUV-Vis absorption spectra of BV at different pH levels, illustrating changes in spectral characteristics with pH variation.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ed,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eUV-Vis absorption spectra of BV incubated with Fe²⁺ at different pH levels, showing the impact of metal ion interaction on the spectra.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ee,\u003c/strong\u003e\u003c/em\u003e HPLC chromatograms of BV incubated with Fe²⁺ under the addition of HEDP and pH adjustment, demonstrating the chelation effect on BV stability.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ef,\u003c/strong\u003e\u003c/em\u003e HPLC chromatograms of BR incubated with Fe²⁺ under the addition of HEDP and pH adjustment, highlighting the prevention of degradation and maintaining BR stability.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/144ea6a685edc80662efe786.jpg"},{"id":83612723,"identity":"44547288-3d1a-4e9d-b75a-73bce8077c56","added_by":"auto","created_at":"2025-05-29 12:44:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":161705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of CO's impact on heme coordination and utilization by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRn\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eHO-1\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eΔ22\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eReaction progress from heme to bilirubin following the elimination of Fe²⁺ interference.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eb,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eRelative activity of \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e using CO-pretreated heme as the substrate.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ec,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eVisualization of the CO release channel (light blue) and the substrate-binding pocket in \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e, with hydrophobicity increasing from white to red (indicating higher hydrophobicity).\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ed,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eQuantum mechanics (QM) calculations of the coordination energies of heme with O₂ and CO.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ee,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eBinding energies of \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e with heme-O₂ and heme-CO.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ef,\u003c/strong\u003e\u003c/em\u003e Substrate-binding pocket of \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e (shown as a cartoon loop) complexed with heme-O₂. Ligand and key residue were displayed as sticks with van der Waals surfaces. Residue color (blue to white to red) represents the hindrance to ligand binding, from low to high.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eg,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eSubstrate-binding pocket of \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e (cartoon loop) complexed with heme-CO. Ligand and key residue were displayed as sticks with van der Waals surfaces. Residue color (blue to white to red) represents the hindrance to ligand binding, from low to high.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003eh,\u003c/strong\u003e\u003c/em\u003e Difference in binding energy contributions of individual residues to heme-CO versus heme-O₂. Positive values indicate residues that contribute stronger hindrance to heme-CO binding.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ei,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eFree energy landscape of the heme-O₂–\u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e complex.\u003cbr\u003e\n\u003cem\u003e\u003cstrong\u003ej,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eFree energy landscape of the heme-CO–\u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e complex.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/adf38321d1ddf686e09375d1.jpg"},{"id":83612158,"identity":"4992d7db-022e-4cb7-b3b1-917496125604","added_by":"auto","created_at":"2025-05-29 12:36:11","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":91492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMulti-enzyme cascade system for high-efficiency BR synthesis.\u003c/strong\u003e\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ea,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThe state of the reaction mixture and HPLC chromatogram before the reaction (initial heme concentration: 1953 mg/L).\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003eb,\u003c/strong\u003e\u003c/em\u003e Schematic diagram of the multi-enzyme cascade catalysis process.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ec,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThe state of the reaction mixture and HPLC chromatogram after the reaction.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ed,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eOverview of the multi-enzyme cascade process.\u003cbr\u003e\n \u003cem\u003e\u003cstrong\u003ee,\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eComparison of different strategies for bilirubin biosynthesis.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/1591115ab3ae95c7b12ac3d1.jpg"},{"id":101389691,"identity":"fd32c6d2-c415-467f-8eb5-059248c39151","added_by":"auto","created_at":"2026-01-29 08:07:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2354473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/216c5e15-5692-490e-af06-c2cbdc761636.pdf"},{"id":83612163,"identity":"7ed61d01-24e6-471d-933f-4569820619d3","added_by":"auto","created_at":"2025-05-29 12:36:12","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":6606705,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6554107/v1/397d1e15c3f99661240738ce.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mechanistic Multi-Enzyme Engineering for High-Yield Bilirubin Biosynthesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBilirubin (BR), the principal bile pigment, was historically considered a metabolic waste product due to its pathological accumulation in conditions such as kernicterus and neonatal jaundice\u003csup\u003e1-3\u003c/sup\u003e. However, recent studies have revealed its beneficial physiological roles. BR possesses potent antioxidant activity, protecting lipids, proteins, and other biomolecules from oxidative stress⁴. Moderate serum BR levels are associated with reduced risks of cardiovascular and metabolic diseases³, and BR exerts neuroprotective effects by mitigating neuronal damage in the central nervous system⁵. Its anti-inflammatory, antioxidant, and antiviral properties have also been investigated in the context of COVID-19 treatment⁶. Furthermore, BR is the primary active compound in \u003cem\u003eCalculus bovis\u003c/em\u003e, a traditional Chinese medicine with significant market value in China⁷.\u003c/p\u003e\n\u003cp\u003eAlthough BR is abundant in animal bile, conventional extraction from porcine or bovine sources is inefficient and cost-prohibitive, yielding only ~300 mg of BR per liter of bile⁸. Synthetic biology approaches have enabled enzymatic and \u003cem\u003ede novo\u003c/em\u003e biosynthesis of tetrapyrrole compounds, including BR⁹\u003csup\u003e,\u003c/sup\u003e¹⁰. In nature, BR is derived from heme degradation: heme oxygenase (HO, EC 1.14.14.18) oxidizes heme to biliverdin (BV), which is subsequently reduced to BR by NADPH-dependent biliverdin reductase (BVR, EC 1.3.1.24)¹¹\u003csup\u003e–\u003c/sup\u003e¹⁵ (Fig. 1a). HO isoforms exhibit regioselectivity by cleaving specific methylene bridges on the porphyrin ring to produce BV isomers (IXα–γ), but most BVRs selectively convert BV IXα into BR IXα—the biologically active form referred to hereafter as BR.\u003c/p\u003e\n\u003cp\u003eHO-mediated heme degradation requires three equivalents of O₂ and seven equivalents of NADPH, but HO lacks intrinsic cofactor-binding domains. Instead, electron transfer is facilitated by redox partner proteins (RPs), which typically consist of a large NADPH/FAD-binding domain and a smaller FMN-binding domain¹⁶\u003csup\u003e–\u003c/sup\u003e¹⁸. In some organisms, these domains are distributed across separate proteins and use Fe–S clusters to shuttle electrons¹⁹\u003csup\u003e–\u003c/sup\u003e²¹ (Fig. 1d). RP conformational changes regulate their activity: the closed conformation transfers electrons from NADPH to FMN, while the open conformation enables FMN to reduce the heme-bound HO²² (Fig. 1b). Subsequent O₂ binding and activation trigger porphyrin ring cleavage, generating BV, Fe²⁺, and carbon monoxide (CO) as byproducts (Fig. 1c, Supplementary Fig. 1)¹⁶.\u003c/p\u003e\n\u003cp\u003eBVR is a nicotinamide-dependent oxidoreductase with a Rossmann-fold N-terminal domain²³\u003csup\u003e,\u003c/sup\u003e²⁴. Structural studies have revealed that two BV molecules bind in the BVR active site; the distal BV may serve as an electron relay during the reduction of the proximal BV to BR²⁴ (Fig. 1e and 1f; Supplementary Fig. 2). \u003cem\u003eDe novo\u003c/em\u003e heme biosynthesis via metabolic engineering has been widely explored²⁵\u003csup\u003e–\u003c/sup\u003e²⁸, and redirection of metabolic flux toward BR using HO and BVR has been attempted²⁹. However, the complexity of the biosynthetic pathway has limited yields, with the highest reported BR titer at just 75.5 mg/L³⁰. Alternatively, a two-step enzymatic conversion of heme to BR holds greater promise. Although diverse HO sources have been explored—including plant³¹, bacterial³², and cyanobacterial³³ enzymes—BV titers remain modest (up to 132 mg/L)³¹, and BVR-mediated BR production has reached only 325 mg/L³⁴. Notably, one-pot enzymatic synthesis of BR from heme has not yet been realized.\u003c/p\u003e\n\u003cp\u003eImportantly, the tetrapyrrolic structures of BV and BR retain metal-chelating properties similar to heme\u003csup\u003e35\u003c/sup\u003e. Transition metal complexes such as BV–Cu²⁺ are prone to oxidative degradation³⁶\u003csup\u003e,\u003c/sup\u003e³⁷, and BR can form Ca²⁺ and Cu²⁺ complexes that generate reactive oxygen species (ROS), leading to its breakdown³⁸\u003csup\u003e,\u003c/sup\u003e³⁹. In particular, Fe²⁺, a byproduct of HO activity, may compromise the structural integrity of both BV and BR. Additionally, CO—another byproduct—has strong ligand-binding properties. Its polarized bond and high electron density at the carbon center allow it to outcompete O₂ for coordination with heme iron⁴⁰. CO binding stabilizes heme–CO complexes, which inhibit the formation of O₂-activated catalytic intermediates, thus preventing substrate access and further enzymatic turnover. These mechanistic insights suggest that uncontrolled byproduct interactions may be a critical factor limiting BR biosynthesis yields, which currently remain in the milligram per liter range.\u003c/p\u003e\n\u003cp\u003eIn this study, we constructed a compact enzyme library through phylogenetic analysis to identify high-activity variants of HO, RP, and BVR, which were heterologously expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3). Truncation of hydrophobic regions improved the soluble expression of HO and RP. To overcome the biosynthetic bottlenecks, we employed computational modeling to elucidate key mechanisms of byproduct interference. Density functional theory (DFT) simulations revealed Fe²⁺-induced O₂ activation and radical-driven oxidative degradation of BV/BR. We also demonstrated that CO-heme binding substantially increases the free energy of heme–CO complexes, inhibiting formation of O₂-coordinated intermediates and blocking substrate access to the enzyme active site. Guided by these insights, we developed an optimized bilirubin biosynthesis system that integrates metal chelation, protonation state control, and enzymatic CO elimination. This strategy enabled gram-scale BR production with the highest yield reported to date, offering a robust platform for industrial-scale synthesis.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eScreening of key enzymes HO, RP, and BVR for construction of the BR biosynthetic pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMammals and plants typically harbor two heme oxygenase isoforms\u0026mdash;inducible HO-1 and constitutive HO-2⁴\u0026sup1;\u0026mdash;which share high sequence and structural similarity, along with a conserved catalytic mechanism. In contrast, microorganisms usually encode a single HO isoform capable of synthesizing BR IX\u0026alpha;, while some bacteria express HugZ-family heme-degrading enzymes that generate alternative isomers such as BR IX\u0026beta; and BR IX\u0026delta;⁴\u0026sup2;. Evolutionary divergence is also reflected in the redox partner proteins (RPs) that support HO activity. In animals and fungi, RPs are generally NADPH-cytochrome P450 oxidoreductases (CPRs; EC 1.6.2.4)⁴\u0026sup3;. By contrast, bacterial and plant systems utilize two discrete proteins for electron transfer: the first protein\u0026mdash;either a ferredoxin (FDX) harboring Fe\u0026ndash;S clusters or a flavodoxin containing FMN\u0026mdash;accepts electrons from NADPH, and the second\u0026mdash;ferredoxin-NADP⁺ reductase or flavodoxin-NADP⁺ reductase (FDR)\u0026mdash;completes the transfer\u003csup\u003e2\u003c/sup\u003e⁰\u003csup\u003e,2\u003c/sup\u003e\u0026sup1;.\u003c/p\u003e\n\u003cp\u003eBiliverdin reductase (BVR), which catalyzes the conversion of BV to BR, is broadly distributed among animals and bacteria. In animals, BVRs are classified into BVR-A and BVR-B subfamilies, distinguished by regioselectivity⁴⁴\u003csup\u003e,\u003c/sup\u003e⁴⁵. Bacterial BVRs predominantly generate BR IX\u0026alpha;, although some rare variants utilize F₄₂₀H₂ as a cofactor in place of NADPH⁴⁶.\u003c/p\u003e\n\u003cp\u003eTo identify the most effective enzyme combinations for BR biosynthesis, we performed phylogenetic analysis of HO (Fig. 2a), RP (Fig. 2b), and BVR (Fig. 2c), selecting evolutionarily diverse candidates to maximize functional variability while maintaining representation across known classes. Representative enzymes were heterologously expressed in \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) (Supplementary Fig. 3), purified, and subjected to activity screening.\u003c/p\u003e\n\u003cp\u003eFor the heme-to-BV conversion step, we tested various HO-RP pairings. Among these, \u003cem\u003eRattus norvegicus\u003c/em\u003e HO-1 (\u003cem\u003eRn\u003c/em\u003eHO-1) in combination with its native CPR (\u003cem\u003eRn\u003c/em\u003eCPR) yielded the highest activity, producing 53.2 mg/L BV within 10 minutes (Fig. 2d). For the subsequent BV-to-BR step, the BVR-A also from \u003cem\u003eRattus norvegicus\u003c/em\u003e (\u003cem\u003eRn\u003c/em\u003eBVR-A, hereafter referred to as \u003cem\u003eRn\u003c/em\u003eBVR) exhibited the most robust catalytic performance, generating 338 mg/L BR in 10 minutes (Fig. 2e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTerminal truncation of HO and CPR enhances soluble expression without compromising catalytic efficiency\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoluble expression of \u003cem\u003eRn\u003c/em\u003eHO-1 and \u003cem\u003eRn\u003c/em\u003eCPR in \u003cem\u003eE. coli\u003c/em\u003e was initially limited, consistent with the presence of predicted membrane-associating domains. Structural analysis revealed that the N-terminal residues 1\u0026ndash;54 of \u003cem\u003eRn\u003c/em\u003eCPR and the C-terminal residues 268\u0026ndash;289 of \u003cem\u003eRn\u003c/em\u003eHO-1 comprise highly hydrophobic regions (Fig. 3a), previously implicated in anchoring these proteins to the endoplasmic reticulum membrane⁴⁷. To enhance expression, we implemented a stepwise truncation strategy to remove these regions. Soluble expression levels increased with the extent of truncation, with the fully truncated variants \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e and \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003e\u0026Delta;54\u003c/sub\u003e exhibiting the highest yields (Fig. 3b).\u003c/p\u003e\n\u003cp\u003eTo confirm that truncation did not impair enzyme function, we first optimized the \u003cem\u003eRn\u003c/em\u003eCPR-to-\u003cem\u003eRn\u003c/em\u003eHO-1 ratio for maximal catalytic activity (Supplementary Fig. 4), then evaluated the kinetic properties of the truncated enzyme pairs. All \u003cem\u003eRn\u003c/em\u003eHO-1 and \u003cem\u003eRn\u003c/em\u003eCPR truncation combinations retained comparable heme affinity (\u003cem\u003eK\u003c/em\u003eₘ) and catalytic efficiency (\u003cem\u003ek\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003eₐₜ/\u003cem\u003eK\u003c/em\u003eₘ) relative to their full-length counterparts (Fig. 3c and Supplementary Fig. 5), indicating that removal of membrane-associated domains did not compromise catalytic function. By contrast, strong substrate inhibition observed in the downstream bilirubin reductase (\u003cem\u003eRn\u003c/em\u003eBVR) reaction precluded accurate kinetic modeling (Supplementary Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLow yield in cascade processes: An unexpected phenomenon\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite the use of cell lysates with equal concentrations of catalysts, the combination of \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e and \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003e\u0026Delta;54\u003c/sub\u003e exhibited a significantly faster reaction rate than the wild-type enzyme combination. However, an unexpected deviation from stoichiometric predictions was observed: while heme consumption and biliverdin (BV) accumulation were expected to be directly proportional, at 80% substrate conversion, BV yield only reached approximately 50%, with BV levels declining sharply as substrate consumption slowed (Fig. 4a). Notably, this issue was absent in the BVR-catalyzed BV-to-BR conversion, where reaction kinetics aligned with expectations (Fig. 4b).\u003c/p\u003e\n\u003cp\u003eThe faster reaction rate of \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e/\u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003e\u0026Delta;54\u003c/sub\u003e suggested a high catalytic activity for the heme-to-BV step; however, the observed discrepancy in BV yield points to an additional factor influencing the reaction. Given the significantly higher specific activity of \u003cem\u003eRn\u003c/em\u003eBVR (Fig. 3c), which catalyzed the subsequent BV-to-BR conversion, we hypothesized that the slower first step of the cascade (heme-to-BV) might be the bottleneck. To optimize reaction conditions and better balance cascade kinetics, we refined the experimental setup (Supplementary Fig. 7).\u003c/p\u003e\n\u003cp\u003eAfter optimizing conditions, we performed one-pot BR synthesis from heme. While the conversion rate of heme increased to 83.9% after 150 minutes, the BR yield plateaued at 48.1% (Fig. 4c). This low yield could not be attributed to external factors, such as UV irradiation\u003csup\u003e48\u003c/sup\u003e or ROS\u003csup\u003e49\u003c/sup\u003e (e.g., H₂O₂), which are known to cause degradation of BR into byproducts like propentdyopents, as our reactions were conducted in the dark and without exogenous ROS. In contrast, the \u003cem\u003eRn\u003c/em\u003eBVR-catalyzed reaction alone performed as expected, suggesting that the issue lies in the earlier heme-to-BV conversion step. We hypothesize that byproducts generated during heme-to-BV conversion, such as Fe\u0026sup2;⁺ or CO, may destabilize BV or BR, thereby limiting the final BR yield.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnveiling the mechanism of Fe\u0026sup2;⁺-enhanced product reactivity and oxidative degradation promotion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first explored the role of Fe\u0026sup2;⁺, a byproduct of HO-catalyzed heme degradation, in promoting the reactivity and oxidative degradation of bilirubin (BR) and its intermediate biliverdin (BV). When standard solutions of BV and BR were incubated with Fe\u0026sup2;⁺ under enzymatic reaction conditions (35\u0026deg;C, pH 7.5) for 150 minutes, HPLC analysis revealed the emergence of multiple unknown compounds (Fig. 5a and 5b), alongside a significant alteration in the UV-Vis spectra (Supplementary Fig. 8), which corresponded with a reduction in the peak areas of BV and BR. LC-MS analysis identified 13 potential degradation products (Supplementary Figs. 9-11).\u003c/p\u003e\n\u003cp\u003eGiven the established coordination chemistry between porphyrins and metal ions, we hypothesized that Fe\u0026sup2;⁺ initially coordinates with BV/BR, thereby triggering degradation. DFT-based electrostatic potential surface analysis suggested that deprotonation of the central nitrogen atoms in BV and BR reduces their local electrostatic potential, enhancing Fe\u0026sup2;⁺ coordination (Supplementary Fig. 12a). Time-dependent DFT (TD-DFT) calculations further indicated that deprotonation of BV induces red shifts in the Soret (~380 nm) and Q bands (690 nm), with a noticeable amplification in Q-band intensity (Supplementary Fig. 12b and 12c), consistent with the experimental UV-Vis changes observed (Fig. 6c). These findings confirm that Fe\u0026sup2;⁺ preferentially interacts with deprotonated BV/BR (denoted as deBV/deBR), aligning with prior studies on porphyrin-metal ion coordination\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eDFT calculations revealed favorable coordination energies for deBV-Fe\u0026sup2;⁺ (-126.5 kcal/mol) and deBR-Fe\u0026sup2;⁺ (-133.5 kcal/mol) (Fig. 5c). Notably, upon Fe\u0026sup2;⁺ binding, deBR transitions from a saddle-shaped to a distorted planar conformation. LC-MS confirmed Fe\u0026sup2;⁺-mediated oxidative degradation of BV and BR without the need for exogenous reactive oxygen species (ROS). We propose that Fe\u0026sup2;⁺ acts as a coordination core, recruiting axial ligands such as O₂ or OH⁻ from the solvent, which in turn enhances the reactivity of the entire complex. To explore this hypothesis further, we investigated various possible axial coordination modes (Supplementary Figs. 13 and 14). Among these, deBV-Fe-OH⁻ exhibited the lowest coordination energy, but OH⁻ ligands poorly attack the porphyrin ring. Therefore, we identified plausible coordination modes, including deBV-Fe-O₂-OH (-9.5 kcal/mol) and deBR-Fe-O₂ (-27.6 kcal/mol) (Fig. 5d).\u003c/p\u003e\n\u003cp\u003eFrontier molecular orbital (FMO) analysis revealed that Fe\u0026sup2;⁺ coordination minimally alters the HOMO/LUMO distributions on the porphyrin ring (Fig. 5e and 5f). However, axial ligand binding (such as O₂) reduces the LUMO electron density on the porphyrin ring while shifting LUMO localization toward the O₂ ligand. Notably, Fe\u0026sup2;⁺ coordination has minimal impact on the overall HOMO-LUMO gap, but the introduction of axial ligands significantly narrows the gap (from 2.07 eV to 0.56 eV for deBV-Fe-O₂-OH and from 2.83 eV to 0.72 eV for deBR-Fe-O₂). These findings suggest that complexes with axial ligands, such as deBV-Fe-O₂-OH and deBR-Fe-O₂, exhibit enhanced reactivity, rendering them energetically favorable for single-electron transfer.\u003c/p\u003e\n\u003cp\u003eGiven that FMO analysis indicated O₂ may act as an electron acceptor, and combining with other heme enzyme mediated ring opening reaction mechanisms\u003csup\u003e50\u003c/sup\u003e, we propose that this coordination facilitates the formation of superoxide radicals (O₂ \u0026rarr; O₂⁻\u0026bull;), which then trigger a radical cascade leading to \u003cem\u003emeso\u003c/em\u003e-carbon attack and subsequent oxidative degradation (Fig. 5g). The proposed mechanism includes the following steps: (1) direct radical addition of O₂⁻\u0026bull; to \u003cem\u003emeso\u003c/em\u003e-carbon, generating a Fe-O-O-carbon intermediate with a \u0026beta;-carbon radical; (2) homolytic cleavage of the O-O bond, forming epoxide intermediates and causing Fe-O bond cleavage; (3) hydrogen transfer from \u003cem\u003emeso\u003c/em\u003e-carbon to the epoxide ring, resulting in the formation of a \u003cem\u003emeso\u003c/em\u003e-carbon radical; and (4) free radical addition of another superoxide radical to \u003cem\u003emeso\u003c/em\u003e-carbon, inducing a ring-opening reaction and leading to oxidative degradation products. This mechanism positions Fe\u0026sup2;⁺ not just as a passive byproduct of heme degradation, but as an active driver of porphyrin instability, promoting both structural disruption and radical initiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreventing Fe\u0026sup2;⁺-induced degradation through competitive chelation and protonation state modulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo mitigate Fe\u0026sup2;⁺-induced degradation, we tested various chelators. While all chelators preserved the absorption features of BV and BR (Supplementary Fig. 15), tartrate and other chelators inhibited enzyme activity in both HO- and BVR-mediated reactions (Fig. 6a and 6b). Etidronic acid (HEDP) emerged as the most effective chelator, significantly enhancing BV and BR yields by 149.8% and 145.6%, respectively (Fig. 6a and 6b). Acid-base titration and DFT calculations confirmed that HEDP exists as di-/tri-deprotonated species at the enzymatic pH, and coordination titration established a 2:1 ratio of HEDP to Fe\u0026sup2;⁺ (Supplementary Fig. 16). Despite this favorable coordination (\u0026Delta;G = -106.9 kcal/mol, Supplementary Fig. 17), residual degradation products persisted (Fig. 6e and 6f), suggesting that the suppression of deBV/deBR-Fe\u0026sup2;⁺ formation was incomplete.\u003c/p\u003e\n\u003cp\u003eGiven that Fe\u0026sup2;⁺ coordination is pH-dependent, we optimized the pH to further mitigate degradation. While BR precipitated under acidic conditions, BV studies across a pH range of 3.5-9.5 showed progressive red shifts in the Soret and Q bands upon deprotonation (Fig. 6c). Addition of Fe\u0026sup2;⁺ under alkaline conditions drastically reduced BV absorption, whereas acidic pH preserved its spectral integrity (Fig. 6d). Adjusting the pH to mildly acidic conditions, in combination with HEDP addition, effectively reduced degradation (Fig. 6e and 6f). The enzyme activity and overall reaction efficiency were optimized by adjusting the HEDP concentration (15 mM) and pH (6.5), ensuring minimal degradation and enhanced product yields (Supplementary Fig. 18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnother cause of unsatisfactory yield in cascade reactions: CO byproduct inhibits heme utilization by HO enzyme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder optimized HEDP dosage and pH conditions to suppress Fe\u0026sup2;⁺ interference, the BR yield increased significantly to 80.1% (1953 mg/L, 3 mM heme as substrate) with the \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e, \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003e\u0026Delta;54\u003c/sub\u003e, and \u003cem\u003eRn\u003c/em\u003eBVR mixture. However, approximately 20.5% of heme remained unconverted during the late stages of the reaction (Fig. 7a). This phenomenon led us to consider CO poisoning\u003csup\u003e51\u003c/sup\u003e, a well-documented issue where CO, another byproduct of HO catalysis, may act as an inhibitor. CO is generated from the \u003cem\u003emeso\u003c/em\u003e-carbon atom of heme, transported through a narrow gas channel in the enzyme, and subsequently released into the solvent environment (Fig. 7c).\u003c/p\u003e\n\u003cp\u003eDFT calculations revealed that heme-CO coordination (\u0026Delta;G = -40.8 kcal/mol) is thermodynamically favored over heme-O₂ coordination (\u0026Delta;G = -23.3 kcal/mol) (Fig. 7d). Consistently, activity assays with CO-pretreated heme solutions showed minimal enzyme catalysis in \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e (Fig. 7b). These results, in combination with QM calculations indicating strong coordination between heme and both O₂ and CO ligands, suggest that free heme is unlikely to persist in aerobic solvent environments.\u003c/p\u003e\n\u003cp\u003eTo further explore the effects of CO on enzyme activity, we performed molecular dynamics simulations. These simulations revealed distinct binding free energies of -8.53 kcal/mol for heme-O₂ and -4.51 kcal/mol for heme-CO (Fig. 7e), suggesting that the enzyme has a significantly weaker affinity for heme-CO. Per-residue decomposition analysis of the binding free energies identified key residues around the substrate-binding pocket that contribute to this difference (Fig. 7f and 7g). Notably, Ser142, which is located near the CO ligand, exhibited higher energy levels in the heme-CO complex compared to heme-O₂, suggesting substantial steric hindrance. This hindrance arises from the rigid, linear geometry of the sp-hybridized CO ligand, in contrast to the naturally bent sp\u0026sup2;-hybridized O₂ ligand, which adopts an optimal bond angle to minimize spatial conflicts.\u003c/p\u003e\n\u003cp\u003eEnergy differences between heme-CO and heme-O₂ binding configurations across all residues are shown in Fig. 7h. The free energy landscape constructed from the RMSD and radius of gyration (Supplementary Fig. 19) during the stable phase of the simulation revealed a deeper and broader energy well for the heme-O₂-enzyme complex (Fig. 7i) compared to the heme-CO complex (Fig. 7j), further supporting the thermodynamic preference for heme-O₂.\u003c/p\u003e\n\u003cp\u003eOur computational analyses provide a mechanistic explanation for CO-induced inhibition of heme utilization in \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e: the formation of stable heme-CO complexes impedes proper substrate positioning within the catalytic pocket, blocking the subsequent O₂-dependent activation of the reaction intermediate. This competitive coordination mechanism is likely exacerbated by CO\u0026apos;s accumulation in the solvent environment, where it functions as a potent heme-sequestering agent, outcompeting O₂ for coordination and hindering enzyme catalysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEfficient bilirubin synthesis enabled by CO removal and NADPH regeneration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo mitigate CO-mediated inhibition, we introduced an engineered carbon monoxide dehydrogenase (\u003cem\u003eCh\u003c/em\u003eCODH)\u003csup\u003e52\u003c/sup\u003e to oxidize CO into non-toxic CO₂. Additionally, to enable cofactor regeneration, we incorporated an engineered NADPH-dependent formate dehydrogenase (\u003cem\u003eAp\u003c/em\u003eFDH)\u003csup\u003e53\u003c/sup\u003e. For multi-enzyme co-expression, we used pCDFDuet-1 and pETDuet-1 plasmids, adding an extra ribosome binding site (RBS) on pETDuet-1. This strategy led to the construction of a five-enzyme co-expression strain that co-expressed \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003e\u0026Delta;22\u003c/sub\u003e, \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003e\u0026Delta;54\u003c/sub\u003e, \u003cem\u003eRn\u003c/em\u003eBVR, \u003cem\u003eAp\u003c/em\u003eFDH, and \u003cem\u003eCh\u003c/em\u003eCODH (Supplementary Fig. 20). A schematic of the multi-enzyme cascade catalysis system is shown in Fig. 8b.\u003c/p\u003e\n\u003cp\u003eThis optimized cascade system successfully converted heme to bilirubin (BR), achieving complete conversion of 1953 mg/L (3 mM) heme to 1678.4 mg/L (2.87 mM) BR, resulting in a 95.8% yield in a 1 L reaction system (Fig. 8d). A color transition from dark brown to reddish-brown was observed throughout the process (Fig. 8a, 8c). BR was extracted from the reaction mixture via chloroform extraction, followed by rotary evaporation and lyophilization. The resulting powder was analyzed by NMR spectroscopy, with the \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra confirming that the synthesized BR adopts the IX\u0026alpha; configuration (Supplementary Fig. 21 and 22).\u003c/p\u003e\n\u003cp\u003eIn contrast to previous reports, this study represents the first successful enhancement of biosynthesized bilirubin production to gram-scale quantities (Fig. 8e). While existing research on bilirubin biosynthesis often utilizes readily available carbon sources such as glucose, which offers cost-effectiveness, our optimized system achieved a remarkable 20-fold higher yield compared to previous studies. Furthermore, the rational truncation strategy employed for improved protein expression efficiency, alongside the systematic byproduct elimination protocol developed here, provides valuable insights for metabolic engineering strategies aimed at optimizing bilirubin biosynthesis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study redefines the enzymatic synthesis of BR by addressing two long-standing yet previously uncharacterized bottlenecks—Fe²⁺-mediated oxidative degradation and CO-induced enzyme inhibition—while achieving record-breaking production titers. The insights and strategies developed herein not only overcome critical limitations in BR biosynthesis but also provide a broadly applicable blueprint for optimizing complex enzymatic cascades in synthetic biology and industrial biocatalysis.\u003c/p\u003e\n\u003cp\u003eA central contribution lies in the mechanistic elucidation of Fe²⁺-induced product instability, a phenomenon not previously recognized in BR synthesis systems. Earlier studies attributed low yields to enzymatic inefficiency or substrate inhibition, overlooking the role of byproduct-metal interactions. Through DFT and TD-DFT\u0026nbsp;analyses, we demonstrated that Fe²⁺ coordinates with deprotonated biliverdin (deBV) and bilirubin (deBR), forming reactive intermediates prone to radical-driven oxidative degradation (Fig. 5). This finding challenges the conventional view of Fe²⁺ as a benign byproduct and establishes metal chelation as an essential strategy for stabilizing tetrapyrrole biosynthesis. The combined use of etidronic acid (HEDP) and pH modulation to suppress deprotonation offers a novel, universally applicable approach to mitigating metal-mediated degradation in porphyrin-based pathways—a strategy previously absent for BR production efforts.\u003c/p\u003e\n\u003cp\u003eEqually significant is the identification of CO as a catalytic poison in heme oxygenase (HO)-mediated systems. While CO’s affinity for heme is well-documented in respiratory biology, its inhibitory effect on HO catalysis had not been mechanistically dissected.\u0026nbsp;Our molecular dynamics simulations revealed that CO’s linear geometry induces steric clashes with key residues (e.g., Ser142), thereby hindering substrate access to the HO active site. This discovery has broad implications for heme-dependent enzymes beyond BR synthesis. The incorporation of \u003cem\u003eCh\u003c/em\u003eCODH to actively eliminate CO represents the first successful application of gas-phase byproduct removal in BR biosynthesis and offers a scalable solution to a long-standing but overlooked issue in gas-ligand and heme-containing enzymatic systems.\u003c/p\u003e\n\u003cp\u003eThe yield achieved in this study—1,678 mg/L BR at 95.8% yield—marks a paradigm shift in enzymatic BR production. Comparation with previous reports underscores this advance: earlier enzymatic systems peaked at 325 mg/L\u003csup\u003e34\u003c/sup\u003e, while \u003cem\u003ede novo\u003c/em\u003e microbial approaches struggled to exceed 75.5 mg/L\u003csup\u003e30\u003c/sup\u003e. This 20-fold enhancement stems from addressing dual byproduct interferences—a factor neglected in prior efforts that focused on enzyme overexpression or cofactor supplementation\u003csup\u003e29,31,33\u003c/sup\u003e. For instance, while Liu et al.\u003csup\u003e29\u003c/sup\u003e attributed yield limitations to HO-1 kinetics, our work demonstrates that even optimized oxygenase activity cannot overcome unchecked Fe²⁺- and CO-mediated losses. These comparisons highlight the necessity of holistic pathway optimization that considers both enzymatic and non-enzymatic factors.\u003c/p\u003e\n\u003cp\u003eBeyond BR synthesis, this study offers methodological innovations with cross-disciplinary relevance. The integration of DFT-guided mechanistic prediction and enzymatic byproduct management exemplifies a \"design-test-learn\" strategy applicable to other metalloenzyme vulnerable to metal toxicity or gas-phase inhibition. For example, cytochrome P450 cascades, which also generate CO and reactive metals\u003csup\u003e55\u003c/sup\u003e, could benefit from \u003cem\u003eCh\u003c/em\u003eCODH or targeted chelators to enhance stability and performance. Furthermore, the rational truncation of membrane-associated domains in \u003cem\u003eRn\u003c/em\u003eHO-1 and \u003cem\u003eRn\u003c/em\u003eCPR provides a practical strategy for solubilizing structurally challenging oxidoreductases, a persistent bottleneck in industrial enzymology\u003csup\u003e56\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this work transcends incremental advances by unraveling and resolving two previously \"invisible\" mechanisms constrained BR biosynthesis. By shifting the focus from isolated enzyme engineering to systemic byproduct management, we establish a new paradigm for pathway optimization—one that improves catalytic efficiency with chemical robustness. These findings not only enable gram-scale BR production but also redefine the boundaries of synthetic biology, proving that computational mechanistic insights and enzymatic innovation can synergistically unlock industrial-scale synthesis of high-value natural products.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll wild-type gene sequences were codon-optimized, synthesized, and cloned into the \u003cem\u003eBam\u003c/em\u003eH I/\u003cem\u003eXho\u0026nbsp;\u003c/em\u003eI restriction sites of the pET28a(+) vector by Sangon Biotech (Shanghai, China). PrimeSTAR Max DNA Polymerase and the restriction enzyme \u003cem\u003eDpn\u0026nbsp;\u003c/em\u003eI were obtained from Takara Bio Inc. (Japan). The homologous recombination and DNA purification kits were purchased from Vazyme Biotech Co., Ltd. (China). The SDS-PAGE gel preparation kit was sourced from Sangon Biotech (Shanghai, China). NADPH was acquired from Roche Holding AG (Switzerland), and heme (hemin chloride, molecular weight = 651.94 Da) was purchased from Aladdin Industrial Corporation (China). Plasmids pETDuet-1, pCDFDuet-1, and \u003cem\u003eE. coli\u003c/em\u003e BL21 (DE3) were preserved in our laboratory stock. All other reagents were sourced from Shanghai Macklin Biochemical Co., Ltd. (China), unless otherwise specified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhylogenetic trees were constructed using the Neighbor–Joining method in Molecular Evolutionary Genetics Analysis version 11 (MEGA11)\u003csup\u003e57\u003c/sup\u003e. Protein sequences used for tree construction were retrieved from the NCBI database, with selected sequences (Supplementary Table 1) representing diverse biological origins and catalytic properties. The tree was visualized using the Chiplot online platform\u003csup\u003e58\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant plasmids were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) strains for protein expression. \u003cem\u003eE. coli\u003c/em\u003e cultures were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) with kanamycin or ampicillin (50 μg/mL) at 37°C with shaking (200 rpm). Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM when the OD\u003csub\u003e600\u003c/sub\u003e reached 0.4‒0.6. After 14 hours of induction at 20°C, cells were collected by centrifugation (8,000 × \u003cem\u003eg\u003c/em\u003e), and SDS-PAGE analysis was performed to verify protein expression levels. For enzyme purification, cells were resuspended in 100 mM Tris-HCl buffer (pH 7.5) and disrupted by ultrasonication on ice. The supernatant was obtained by centrifugation (12,000 × \u003cem\u003eg\u003c/em\u003e for 10 min at 4°C) and purified using Ni²⁺ affinity chromatography, as previously described\u003csup\u003e59\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReactions for HO and its partner proteins CPR or FDR were conducted by adding 100 mg/L HO and either 100 mg/L CPR or 100 mg/L FDR, with 1 mM heme and 10 mM NADPH, followed by incubation at 35°C with agitation (600 rpm) for 1 hour. Reactions were quenched by a 10-fold dilution with DMSO, and product concentrations were analyzed by HPLC. For BVR screening, reactions containing 100 mg/L BVR, 1 mM BV, and 2 mM NADPH were conducted at 35°C with agitation (600 rpm) for 30 minutes, followed by quenching with DMSO dilution, and product levels were determined by HPLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of truncated mutants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTruncated plasmids were generated by amplifying gene sequences with the primer pairs listed in Supplementary Table 2, using pET28a(+)-\u003cem\u003eRn\u003c/em\u003eHO-1 or pET28a(+)-\u003cem\u003eRn\u003c/em\u003eCPR as templates,\u0026nbsp;following a previously reported PCR protocol\u003csup\u003e60\u003c/sup\u003e. The PCR products were treated with \u003cem\u003eDpn\u0026nbsp;\u003c/em\u003eI to remove template plasmids, purified, and subjected to homologous recombination using a Vazyme Biotech kit. The resulting plasmids were transformed into \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eBL21 (DE3), and colonies were screened for the correct mutants, which were validated by DNA sequencing (Sangon Biotech, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLC-MS analysis of BV and BR degradation products was performed using a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer. Chromatographic separation was achieved using a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm particle size; Waters Corporation, Milford, MA, USA) with electrospray ionization (ESI) in positive ion mode. The gradient elution program was as follows: 0‒5 min: 20% B; 5‒10 min: 20%‒40% B; 10‒15 min: 40% B; 15‒20 min: 40%‒100% B; 20‒30 min: 100% B; 30‒32.5 min: 100%‒20% B; 32.5‒35 min: 20% B. The column temperature was maintained at 30°C, and the flow rate was 0.2 mL/min. Data were processed using Xcalibur software (Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of chelators\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor initial chelator screening, 1 mM BV or BR standard solutions were mixed with 5 mM chelator candidates (tartrate, citrate, EDTA, DEG, HEDP, NTA, HEDTA) and 1 mM FeCl₂. After incubation at 35°C for 150 minutes, samples were analyzed by UV-Vis spectroscopy (Supplementary Methods). To evaluate chelator effects on enzymatic activity, crude enzyme reactions were conducted with \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e, \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003eΔ54\u003c/sub\u003e, or \u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e, \u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003eΔ54\u003c/sub\u003e, and \u003cem\u003eRn\u003c/em\u003eBVR in the presence of 5 mM of each chelator. Reactions were performed under conditions (1 mM heme, 10 mM NADPH, 100 mM Tris-HCl, pH 7.5, 35°C, 600 rpm), and products were quantified by HPLC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTheoretical calculations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDensity functional theory (DFT) calculations were performed using Gaussian 09, Revision D.01\u003csup\u003e61\u003c/sup\u003e. Initial molecular structures were modeled in GaussView 5.0 and subjected to conformational searches with the Molclus program\u003csup\u003e62\u003c/sup\u003e. Geometry optimizations and vibrational frequency analyses were performed at the B3LYP/6-311G(d,p) level of theory, and single-point energy calculations were carried out at the BP86/def2-TZVP level. A polarizable continuum model (PCM) was used to simulate aqueous solvation effects, and Grimme’s GD3BJ dispersion correction was applied. For Fe²⁺-containing systems, all possible spin multiplicities were evaluated to determine the ground-state configuration. UV-Vis absorption spectra for BV were calculated using time-dependent DFT (TD-DFT) at the BP86/def2-TZVP level, with 50 excited states computed. Electrostatic potential (ESP) charges and frontier molecular orbitals (FMOs) were analyzed using the Multiwfn program (Version 3.8)\u003csup\u003e63\u003c/sup\u003e, and results were visualized using VMD software (Version 1.9.3)\u003csup\u003e64\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScaled-up BR synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor scaled-up BR synthesis, the reaction was conducted in a 5 L bioreactor (T\u0026amp;J Bioengineering, Shanghai, China). The system contained 20 g dry cell weight (DCW)/L of lysate from the five-enzyme co-expression strain \u003cem\u003eE. coli\u003c/em\u003e/pCDFDuet-1-\u003cem\u003eRn\u003c/em\u003eCPR\u003csub\u003eΔ54\u003c/sub\u003e/\u003cem\u003eRn\u003c/em\u003eHO-1\u003csub\u003eΔ22\u003c/sub\u003e/pETDuet-1-\u003cem\u003eCh\u003c/em\u003eCODH/\u003cem\u003eRn\u003c/em\u003eBVR/\u003cem\u003eAp\u003c/em\u003eFDH, 1953 mg/L (3 mM) heme, 50 mM ammonium formate, 0.1 mM FMN, and 15 mM HEDP. The reaction was conducted at 35°C with agitation (400 rpm), with pH automatically maintained at 6.5 ± 0.1 by controlled addition of HCl, and air was continuously supplied at 2 vvm (volume per volume per minute).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach experiment was performed at least in triplicates. Values were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using Origin2024 with student’s t-test. And a value of p \u0026lt; 0.05 was considered statistically significant.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is included in the main text or Supplementary Information file. Source data are provided with this paper.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors greatly appreciate the financial support from the National Key Research and Development Program of China (2023YFA0914500), the National Science Foundation of China (32271487), the National First-class Discipline Program of Light Industry Technology and Engineering (LITE201812), and the Program of Introducing Talents of Discipline to Universities (111-2-06).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.T.J. and J.X.R. contributed equally to this work. Z.T.J. designed the experiments, analyzed the data, conducted DFT calculation and wrote the first draft. J.X.R. contributed to molecular dynamics simulation, analysis of simulation results and energy calculation. C.K.Z contributed to cell fermentation and protein purification. Y.M.L contributed to generation of mutants. Q.Z contributed to the process of scale-up reaction. W.C.Z. and M.Y.Z. reviewed the manuscript and gave the proferssional suggestions. R.Z.Z. guided the project and revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTell, G. \u0026amp; Gustincich, S. Redox state, oxidative stress, and molecular mechanisms of protective and toxic effects of bilirubin on cells. \u003cem\u003eCurr. Pharm. Design\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 2908-2914 (2009). https://doi.org:10.2174/138161209789058174\u003c/li\u003e\n\u003cli\u003eDu, L.\u003cem\u003e et al.\u003c/em\u003e Neonatal hyperbilirubinemia management: Clinical assessment of bilirubin production. \u003cem\u003eSemin. Perinatol.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 151351 (2021). https://doi.org:10.1016/j.semperi.2020.151351\u003c/li\u003e\n\u003cli\u003eHinds, T. D., Jr. \u0026amp; Stec, D. E. Bilirubin safeguards cardiorenal and metabolic diseases: a protective role in health. \u003cem\u003eCurr. Hypertens. Rep.\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 87 (2019). https://doi.org:10.1007/s11906-019-0994-z\u003c/li\u003e\n\u003cli\u003eZiberna, L., Martelanc, M., Franko, M. \u0026amp; Passamonti, S. Bilirubin is an endogenous antioxidant in human vascular endothelial cells. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 29240 (2016). https://doi.org:10.1038/srep29240\u003c/li\u003e\n\u003cli\u003eJayanti, S., Vitek, L., Tiribelli, C. \u0026amp; Gazzin, S. The Role of bilirubin and the other \u0026quot;yellow players\u0026quot; in neurodegenerative diseases. \u003cem\u003eAntioxidants\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 900 (2020). https://doi.org:10.3390/antiox9090900\u003c/li\u003e\n\u003cli\u003eKhurana, I.\u003cem\u003e et al.\u003c/em\u003e Can bilirubin nanomedicine become a hope for the management of COVID-19? \u003cem\u003eMed. Hypotheses\u003c/em\u003e \u003cstrong\u003e149\u003c/strong\u003e, 110534 (2021). https://doi.org:10.1016/j.mehy.2021.110534\u003c/li\u003e\n\u003cli\u003eYu, Z. J.\u003cem\u003e et al.\u003c/em\u003e Calculus bovis: A review of the traditional usages, origin, chemistry, pharmacological activities and toxicology. \u003cem\u003eJ. Ethnopharmacol.\u003c/em\u003e \u003cstrong\u003e254\u003c/strong\u003e, 112649 (2020). https://doi.org:10.1016/j.jep.2020.112649\u003c/li\u003e\n\u003cli\u003eWang, D. Q. H. \u0026amp; Carey, M. C. Therapeutic uses of animal biles in traditional Chinese medicine: An ethnopharmacological, biophysical chemical and medicinal review. \u003cem\u003eWorld J. Gastroenterol.\u003c/em\u003e \u003cstrong\u003e20\u003c/strong\u003e, 9952-9975 (2014). https://doi.org:10.3748/wjg.v20.i29.9952\u003c/li\u003e\n\u003cli\u003eZhang, J.\u003cem\u003e et al.\u003c/em\u003e Recent advances in microbial production of high-value compounds in the tetrapyrrole biosynthesis pathway. \u003cem\u003eBiotechnol. Adv.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 107904 (2022). https://doi.org:10.1016/j.biotechadv.2021.107904\u003c/li\u003e\n\u003cli\u003eKo, Y. J.\u003cem\u003e et al.\u003c/em\u003e Bioproduction of porphyrins, phycobilins, and their proteins using microbial cell factories: engineering, metabolic regulations, challenges, and perspectives. \u003cem\u003eCrit. Rev. Biotechnol.\u003c/em\u003e, \u003cstrong\u003e44\u003c/strong\u003e, 373-387 (2023). https://doi.org:10.1080/07388551.2023.2168512\u003c/li\u003e\n\u003cli\u003eWaza, A. A., Hamid, Z., Ali, S., Bhat, S. A. \u0026amp; Bhat, M. A. A review on heme oxygenase-1 induction: is it a necessary evil. \u003cem\u003eInflamm. Res.\u003c/em\u003e \u003cstrong\u003e67\u003c/strong\u003e, 579-588 (2018). https://doi.org:10.1007/s00011-018-1151-x\u003c/li\u003e\n\u003cli\u003ePena, A. C. \u0026amp; Pamplona, A. Heme oxygenase-1, carbon monoxide, and malaria-The interplay of chemistry and biology. \u003cem\u003eCoord. Chem. Rev.\u003c/em\u003e \u003cstrong\u003e453\u003c/strong\u003e, 214285 (2022). https://doi.org:10.1016/j.ccr.2021.214285\u003c/li\u003e\n\u003cli\u003ePoulos, T. L. Heme enzyme structure and function. \u003cem\u003eChem. Rev.\u003c/em\u003e \u003cstrong\u003e114\u003c/strong\u003e, 3919-3962 (2014). https://doi.org:10.1021/cr400415k\u003c/li\u003e\n\u003cli\u003eSalim, M., Brown-Kipphut, B. A. \u0026amp; Maines, M. D. Human biliverdin reductase is autophosphorylated, and phosphorylation is required for bilirubin formation. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e276\u003c/strong\u003e, 10929-10934 (2001). https://doi.org:10.1074/jbc.M010753200\u003c/li\u003e\n\u003cli\u003eFlorczyk, U. M., Jozkowicz, A. \u0026amp; Dulak, J. Biliverdin reductase: new features of an old enzyme and its potential therapeutic significance. \u003cem\u003ePharmacol. Rep.\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 38-48 (2008). \u003c/li\u003e\n\u003cli\u003eMatsui, T., Iwasaki, M., Sugiyama, R., Unno, M. \u0026amp; Ikeda-Saito, M. Dioxygen activation for the self-degradation of heme: reaction mechanism and regulation of heme oxygenase. \u003cem\u003eInorg. Chem.\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 3602-3609 (2010). https://doi.org:10.1021/ic901869t\u003c/li\u003e\n\u003cli\u003eSato, H.\u003cem\u003e et al.\u003c/em\u003e Crystal structure of rat haem oxygenase-1 in complex with ferrous verdohaem: presence of a hydrogen-bond network on the distal side. \u003cem\u003eBiochem. J.\u003c/em\u003e \u003cstrong\u003e419\u003c/strong\u003e, 339-345 (2009). https://doi.org:10.1042/BJ20082279\u003c/li\u003e\n\u003cli\u003eHamdane, D.\u003cem\u003e et al.\u003c/em\u003e Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e284\u003c/strong\u003e, 11374-11384 (2009). https://doi.org:10.1074/jbc.M807868200\u003c/li\u003e\n\u003cli\u003eSugishima, M., Wada, K. \u0026amp; Fukuyama, K. Recent advances in the understanding of the reaction chemistries of the heme catabolizing enzymes HO and BVR Based on high resolution protein structures. \u003cem\u003eCurr. Med. Chem.\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e, 3499-3518 (2020). https://doi.org:10.2174/0929867326666181217142715\u003c/li\u003e\n\u003cli\u003eTohda, R.\u003cem\u003e et al.\u003c/em\u003e Crystal structure of higher plant heme oxygenase-1 and its mechanism of interaction with ferredoxin. \u003cem\u003eJ. Biol. Chem.\u003c/em\u003e \u003cstrong\u003e296\u003c/strong\u003e, 100217 (2021). https://doi.org:10.1074/jbc.RA120.016271\u003c/li\u003e\n\u003cli\u003eWang, J.\u003cem\u003e et al.\u003c/em\u003e Enzymological and structural characterization of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e heme oxygenase-1. \u003cem\u003eFEBS Open Bio\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1677-1687 (2022). https://doi.org:10.1002/2211-5463.13453\u003c/li\u003e\n\u003cli\u003eCastrignano, S.\u003cem\u003e et al.\u003c/em\u003e Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops. \u003cem\u003eBiochim. Biophys. Acta Proteins Proteom.\u003c/em\u003e \u003cstrong\u003e1866\u003c/strong\u003e, 116-125 (2018). https://doi.org:10.1016/j.bbapap.2017.07.009\u003c/li\u003e\n\u003cli\u003ePaukovich, N.\u003cem\u003e et al.\u003c/em\u003e Biliverdin reductase B dynamics are coupled to coenzyme binding. \u003cem\u003eJ. Mol. Biol.\u003c/em\u003e \u003cstrong\u003e430\u003c/strong\u003e, 3234-3250 (2018). https://doi.org:10.1016/j.jmb.2018.06.015\u003c/li\u003e\n\u003cli\u003eTakao, H.\u003cem\u003e et al.\u003c/em\u003e A substrate-bound structure of cyanobacterial biliverdin reductase identifies stacked substrates as critical for activity. \u003cem\u003eNat. Commun.\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 14397 (2017). https://doi.org:10.1038/ncomms14397\u003c/li\u003e\n\u003cli\u003eIshchuk, O. P.\u003cem\u003e et al.\u003c/em\u003e Genome-scale modeling drives 70-fold improvement of intracellular heme production in \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, e2108245119 (2022). https://doi.org:10.1073/pnas.2108245119\u003c/li\u003e\n\u003cli\u003eChoi, K. R., Yu, H. E., Lee, H. \u0026amp; Lee, S. Y. Improved production of heme using metabolically engineered \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eBiotechnol. Bioeng.\u003c/em\u003e \u003cstrong\u003e119\u003c/strong\u003e, 3178-3193 (2022). https://doi.org:10.1002/bit.28194\u003c/li\u003e\n\u003cli\u003eChen, H.\u003cem\u003e et al.\u003c/em\u003e High-yield porphyrin production through metabolic engineering and biocatalysis. \u003cem\u003eNat. Biotechnol.\u003c/em\u003e (2024). https://doi.org:10.1038/s41587-024-02267-3\u003c/li\u003e\n\u003cli\u003eYang, S.\u003cem\u003e et al.\u003c/em\u003e Recent advances in microbial synthesis of free heme. \u003cem\u003eAppl. Microbiol. Biotechnol.\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 17-27 (2024). https://doi.org:10.1007/s00253-023-12968-5\u003c/li\u003e\n\u003cli\u003eLiu, Z., Xiong, P., Guo, N. \u0026amp; Chen, H. Efficient biosynthesis of bilirubin by overexpressing heme oxygenase, biliverdin reductase and 5-aminolevulinic acid dehydratase in \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eMol. Catal.\u003c/em\u003e \u003cstrong\u003e571\u003c/strong\u003e, 114714 (2025). https://doi.org:10.1016/j.mcat.2024.114714\u003c/li\u003e\n\u003cli\u003eChen, H., Xiong, P., Guo, N. \u0026amp; Liu, Z. Metabolic engineering of \u003cem\u003eEscherichia coli\u003c/em\u003e for production of a bioactive metabolite of bilirubin. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e (2024). https://doi.org:10.3390/ijms25179741\u003c/li\u003e\n\u003cli\u003eMei, J.\u003cem\u003e et al.\u003c/em\u003e Production of biliverdin by biotransformation of exogenous heme using recombinant \u003cem\u003ePichia pastoris\u003c/em\u003e cells. \u003cem\u003eBioresour. Bioprocess.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 19 (2024). https://doi.org:10.1186/s40643-024-00736-w\u003c/li\u003e\n\u003cli\u003eYan, S.\u003cem\u003e et al.\u003c/em\u003e Efficient production of biliverdin through whole-cell biocatalysis using recombinant \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eChinese J. biotechnol.\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 2581-2593 (2022). https://doi.org:10.13345/j.cjb.220137\u003c/li\u003e\n\u003cli\u003eChen, D., Brown, J. D., Kawasaki, Y., Bommer, J. \u0026amp; Takemoto, J. Y. Scalable production of biliverdin IX alpha by \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eBMC Biotechnol.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 89 (2012). https://doi.org:10.1186/1472-6750-12-89\u003c/li\u003e\n\u003cli\u003eMei, J.\u003cem\u003e et al.\u003c/em\u003e Production of bilirubin by biotransformation of biliverdin using recombinant \u003cem\u003eEscherichia coli\u003c/em\u003e cells. \u003cem\u003eBioprocess. Biosyst. Eng.\u003c/em\u003e \u003cstrong\u003e45\u003c/strong\u003e, 563-571 (2022). https://doi.org:10.1007/s00449-021-02679-4\u003c/li\u003e\n\u003cli\u003eKepp, K. P. Heme: From quantum spin crossover to oxygen manager of life. \u003cem\u003eCoord. Chem. Rev.\u003c/em\u003e \u003cstrong\u003e344\u003c/strong\u003e, 363-374 (2017). https://doi.org:10.1016/j.ccr.2016.08.008\u003c/li\u003e\n\u003cli\u003eSzterenberg, L., Latos‐Grażyński, L. \u0026amp; Wojaczyński, J. Metallobiliverdin radicals\u0026mdash;DFT studies. \u003cem\u003eChemPhysChem\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 691-698 (2003). https://doi.org:10.1002/cphc.200200611\u003c/li\u003e\n\u003cli\u003eDimitrijević, M. S.\u003cem\u003e et al.\u003c/em\u003e Biliverdin\u0026ndash;copper complex at physiological pH. \u003cem\u003eDalton Trans.\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e, 6061-6070 (2019). https://doi.org:10.1039/c8dt04724c\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez, A., L\u0026oacute;pez-Rull, I. \u0026amp; Fargallo, J. A. To prevent oxidative stress, what about protoporphyrin IX, biliverdin, and bilirubin? \u003cem\u003eAntioxidants\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1662 (2023). https://doi.org:10.3390/antiox12091662\u003c/li\u003e\n\u003cli\u003eBozic, B.\u003cem\u003e et al.\u003c/em\u003e Mechanisms of redox interactions of bilirubin with copper and the effects of penicillamine. \u003cem\u003eChem. Biol. Interact.\u003c/em\u003e \u003cstrong\u003e278\u003c/strong\u003e, 129-134 (2017). https://doi.org:10.1016/j.cbi.2017.10.022\u003c/li\u003e\n\u003cli\u003eLiu, S., Xia, S., Yue, D., Sun, H. \u0026amp; Hirao, H. The bonding nature of Fe\u0026ndash;CO complexes in heme proteins. \u003cem\u003eInorg. Chem.\u003c/em\u003e \u003cstrong\u003e61\u003c/strong\u003e, 17494-17504 (2022). https://doi.org:10.1021/acs.inorgchem.2c02387\u003c/li\u003e\n\u003cli\u003eMedina, M. V., Sapochnik, D., Garcia Sola, M. \u0026amp; Coso, O. Regulation of the expression of heme oxygenase-1: Signal transduction, gene promoter activation, and beyond. \u003cem\u003eAntioxid. Redox Signal.\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, 1033-1044 (2020). https://doi.org:10.1089/ars.2019.7991\u003c/li\u003e\n\u003cli\u003eUchida, T., Sekine, Y., Matsui, T., Ikeda-Saito, M. \u0026amp; Ishimori, K. A heme degradation enzyme, HutZ, from \u003cem\u003eVibrio cholerae\u003c/em\u003e. \u003cem\u003eChem. Commun. \u003c/em\u003e\u003cstrong\u003e48\u003c/strong\u003e, 6741-6743 (2012). https://doi.org:10.1039/c2cc31147j\u003c/li\u003e\n\u003cli\u003eSugishima, M.\u003cem\u003e et al.\u003c/em\u003e Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e \u003cstrong\u003e111\u003c/strong\u003e, 2524-2529 (2014). https://doi.org:10.1073/pnas.1322034111\u003c/li\u003e\n\u003cli\u003eDuff, M. R.\u003cem\u003e et al.\u003c/em\u003e Structure, dynamics and function of the evolutionarily changing biliverdin reductase B family. \u003cem\u003eJ. Biochem.\u003c/em\u003e \u003cstrong\u003e168\u003c/strong\u003e, 191-202 (2020). https://doi.org:10.1093/jb/mvaa039\u003c/li\u003e\n\u003cli\u003eO\u0026apos;Brien, L., Hosick, P. A., John, K., Stec, D. E. \u0026amp; Hinds, T. D., Jr. Biliverdin reductase isozymes in metabolism. \u003cem\u003eTrends Endocrinol. Metab.\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 212-220 (2015). https://doi.org:10.1016/j.tem.2015.02.001\u003c/li\u003e\n\u003cli\u003eAhmed, F. H.\u003cem\u003e et al.\u003c/em\u003e Rv2074 is a novel F\u003csub\u003e420\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003e-dependent biliverdin reductase in \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. \u003cem\u003eProtein Sci.\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 1692-1709 (2016). https://doi.org:10.1002/pro.2975\u003c/li\u003e\n\u003cli\u003eRyter, S. W. Heme oxygenase-1: An anti-inflammatory effector in cardiovascular, lung, and related metabolic disorders. \u003cem\u003eAntioxidants\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 555 (2022). https://doi.org:10.3390/antiox11030555\u003c/li\u003e\n\u003cli\u003eStanojevic, J. S., Zvezdanovic, J. B. \u0026amp; Markovic, D. Z. Bilirubin degradation in methanol induced by continuous UV-B irradiation: a UHPLC-ESI-MS study. \u003cem\u003ePharmazie\u003c/em\u003e \u003cstrong\u003e70\u003c/strong\u003e, 225-230 (2015). https://doi.org:10.1691/ph.2015.4122\u003c/li\u003e\n\u003cli\u003eRitter, M.\u003cem\u003e et al.\u003c/em\u003e Isolation and identification of intermediates of the oxidative bilirubin degradation. \u003cem\u003eOrg. Lett.\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, 4432-4435 (2016). https://doi.org:10.1021/acs.orglett.6b02287\u003c/li\u003e\n\u003cli\u003eChung, L. W., Li, X., Sugimoto, H., Shiro, Y. \u0026amp; Morokuma, K. Density functional theory study on a missing piece in understanding of heme chemistry: The reaction mechanism for indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. \u003cem\u003eJ. American Chem. Soc.\u003c/em\u003e \u003cstrong\u003e130\u003c/strong\u003e, 12299-12309 (2008). https://doi.org:10.1021/ja803107w\u003c/li\u003e\n\u003cli\u003eZazzeron, L., Franco, W. \u0026amp; Anderson, R. Carbon monoxide poisoning and phototherapy. \u003cem\u003eNitric Oxide-Biol. Chem.\u003c/em\u003e \u003cstrong\u003e146\u003c/strong\u003e, 31-36 (2024). https://doi.org:10.1016/j.niox.2024.04.001\u003c/li\u003e\n\u003cli\u003eKim, S. M.\u003cem\u003e et al.\u003c/em\u003e O2-tolerant CO dehydrogenase via tunnel redesign for the removal of CO from industrial flue gas. \u003cem\u003eNat. Catal.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 807-817 (2022). https://doi.org:10.1038/s41929-022-00834-y\u003c/li\u003e\n\u003cli\u003eCheng, F.\u003cem\u003e et al.\u003c/em\u003e Switching the cofactor preference of formate dehydrogenase to develop an NADPH-dependent biocatalytic system for synthesizing chiral amino acids. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cstrong\u003e71\u003c/strong\u003e, 9009-9019 (2023). https://doi.org:10.1021/acs.jafc.3c01561\u003c/li\u003e\n\u003cli\u003eZhou, W. \u003cem\u003eet al\u003c/em\u003e. Production of bilirubin via whole-cell transformation utilizing recombinant \u003cem\u003eCorynebacterium glutamicum\u003c/em\u003e expressing a beta-glucuronidase from S\u003cem\u003etaphylococcus sp.\u003c/em\u003e RLH1. \u003cem\u003eBiotechnol. Lett.\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e, 223-233 (2024). https://doi.org:10.1007/s10529-024-03468-1\u003c/li\u003e\n\u003cli\u003eYuan, Z. N., Cruz, L. K. D., Yang, X. X. \u0026amp; Wang, B. H. Carbon monoxide signaling: examining its engagement with various molecular targets in the context of binding affinity, concentration, and biologic responses. \u003cem\u003ePharmacol. Rev.\u003c/em\u003e \u003cstrong\u003e74\u003c/strong\u003e, 823-873 (2022). https://doi.org:10.1124/pharmrev.121.000564\u003c/li\u003e\n\u003cli\u003eKaur, J., Kumar, A. \u0026amp; Kaur, J. Strategies for optimization of heterologous protein expression in \u003cem\u003eE. coli\u003c/em\u003e: Roadblocks and reinforcements. \u003cem\u003eInt. J. Biol. Macromol.\u003c/em\u003e \u003cstrong\u003e106\u003c/strong\u003e, 803-822 (2018). https://doi.org:10.1016/j.ijbiomac.2017.08.080\u003c/li\u003e\n\u003cli\u003eTamura, K., Stecher, G. \u0026amp; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. \u003cem\u003eMol. Biol. Evol.\u003c/em\u003e \u003cstrong\u003e38\u003c/strong\u003e, 3022-3027 (2021). https://doi.org:10.1093/molbev/msab120\u003c/li\u003e\n\u003cli\u003eXie, J.\u003cem\u003e et al.\u003c/em\u003e Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, W587-W592 (2023). https://doi.org:10.1093/nar/gkad359\u003c/li\u003e\n\u003cli\u003eRuan, Y. Q., Zhang, R. Z. \u0026amp; Xu, Y. Directed evolution of maltogenic amylase from \u003cem\u003eBacillus licheniformis\u003c/em\u003e R-53: Enhancing activity and thermostability improves bread quality and extends shelf life. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cstrong\u003e381\u003c/strong\u003e, 132222 (2022). https://doi.org:10.1016/j.foodchem.2022.132222\u003c/li\u003e\n\u003cli\u003eXi, Z. W.\u003cem\u003e et al.\u003c/em\u003e Deciphering the key loop: enhancing l-threonine transaldolase\u0026apos;s catalytic potential. \u003cem\u003eAcs Catal.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 10462-10474 (2024). https://doi.org:10.1021/acscatal.4c02049\u003c/li\u003e\n\u003cli\u003eGaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, and D. J. Fox, Gaussian, Inc., Wallingford CT, 2013.\u003c/li\u003e\n\u003cli\u003eLu T, Molclus program, version 1.12, http://www.keinsci.com/research/molclus.html\u003c/li\u003e\n\u003cli\u003eLu, T. \u0026amp; Chen, F. Multiwfn: a multifunctional wavefunction analyzer. \u003cem\u003eJ. Comput. Chem.\u003c/em\u003e \u003cstrong\u003e33\u003c/strong\u003e, 580-592 (2012). https://doi.org:10.1002/jcc.22885\u003c/li\u003e\n\u003cli\u003eHumphrey, W., Dalke, A. \u0026amp; Schulten, K. VMD: Visual molecular dynamics. \u003cem\u003eJ. Mol. Graph. \u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 33-38 (1996). https://doi.org:10.1016/0263-7855(96)00018-5\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bilirubin, multi-enzyme cascade, radical attack, metal coordination, carbon monoxide inhibition","lastPublishedDoi":"10.21203/rs.3.rs-6554107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6554107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBilirubin biosynthesis has long been limited by low yields and unclear bottlenecks. Here, we report a fully in vitro pathway that coverts heme to bilirubin with the highest reported titer. Through systematically screening and engineering, we identified two hidden challenges: Fe²⁺ causes intermediate degradation, and CO inhibits heme oxygenase activity. Initial yields stalled at 48.1% due to Fe²⁺-induced biliverdin and bilirubin breakdown. We revealed that Fe²⁺ interacts with deprotonated biliverdin and bilirubin, triggering oxidative ring-opening degradation \u003cem\u003evia\u003c/em\u003e O₂-mediated radical mechanism. DFT calculations showed Fe²⁺-ligand complexes reduce the HOMO-LUMO gap, enhancing their elector transfer susceptibility. Competitive chelation of Fe\u003csup\u003e2+\u003c/sup\u003e and protonation-modulation boosted yield to 80.1%. Furthermore, heme-CO complexes block O\u003csub\u003e2\u003c/sub\u003e-activation for accessing heme oxygenase. Introducing carbon monoxide dehydrogenase for CO removal and formate dehydrogenase for NADPH-recycling enabled efficient bilirubin synthesis of 1.7 g/L and 95.8% yield—a 20-fold improvement. Our work shows byproducts control is the key to stabilize heme-related pathways and as an advanced tool in synthetic biology.\u003c/p\u003e","manuscriptTitle":"Mechanistic Multi-Enzyme Engineering for High-Yield Bilirubin Biosynthesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 12:36:07","doi":"10.21203/rs.3.rs-6554107/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e910c19b-0048-41a1-baa3-6ab250eff40c","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48120610,"name":"Biological sciences/Biotechnology/Industrial microbiology"},{"id":48120611,"name":"Biological sciences/Biochemistry/Biocatalysis"},{"id":48120612,"name":"Biological sciences/Biochemistry/Enzymes/Multienzyme complexes"},{"id":48120613,"name":"Biological sciences/Chemical biology/Computational chemistry"}],"tags":[],"updatedAt":"2026-01-29T08:07:19+00:00","versionOfRecord":{"articleIdentity":"rs-6554107","link":"https://doi.org/10.1038/s41467-025-67804-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-23 05:00:00","publishedOnDateReadable":"December 23rd, 2025"},"versionCreatedAt":"2025-05-29 12:36:07","video":"","vorDoi":"10.1038/s41467-025-67804-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67804-3","workflowStages":[]},"version":"v1","identity":"rs-6554107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6554107","identity":"rs-6554107","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}