Biosynthesis of 4-ethylphenol via an enzyme cascade with an engineered fatty acid photodecarboxylase

Biosynthesis of 4-ethylphenol via an enzyme cascade with an engineered fatty acid photodecarboxylase | 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 Research Article Biosynthesis of 4-ethylphenol via an enzyme cascade with an engineered fatty acid photodecarboxylase Zherui Xu, Zhaoyang Qin, Yue Wu, Yi Zhou, Shuke Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7714953/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 4-Ethylphenol (4-EP) is a key platform molecule for the synthesis of flavors, polymers, and pharmaceuticals. The enzyme responsible for its biosynthesis, postulated to be a phloretic acid decarboxylase, has not yet been identified in nature. This study unveils that fatty acid photodecarboxylase (FAP) effectively substitutes for this "missing" enzyme. Upon photoexcitation, the FAP cofactor directly decarboxylates p -hydroxyphenyl propionic acid (HPPA) without prerequisite activation, thereby introducing a novel paradigm of light-driven decarboxylation. A semi-rational design yielded a triple FAP mutant (Y466V/G462V/V453M) with 3.7-fold enhanced activity. Leveraging this discovery, we developed an integrated fermentation-photocatalysis process. Engineered E. coli first produced tyrosine from glucose, followed by a two-step whole-cell biotransformation that achieved a record 4-EP titer of 4.02 mM (0.49 g L⁻¹). This cofactor- and toxin-free route establishes a plug-and-play platform for the sustainable synthesis of aromatic compounds. Biotechnology and Bioengineering biocatalysis enzyme cascades protein engineering photodecarboxylase renewable resources Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Aromatic chemicals are pivotal "functional cores" in diverse sectors—including pharmaceuticals, flavors, cosmetics, food, and advanced materials—owing to their distinct chemical properties. Conventional production, however, depends on petrochemical feedstocks, which is fraught with drawbacks: resource depletion, price instability, high energy costs, and substantial environmental footprint. ( 1 ) Shifting to renewable resources for sustainable synthesis is thus a vital strategic pivot. This transition is being accelerated by synthetic biology and enzyme engineering, which have elevated the microbial production of aromatics from laboratory-scale demonstrations to commercial reality. Key compounds such as vanillin, cinnamic acid, and coumarin are now produced through whole-cell biocatalysis or multi-enzyme systems; crucially, these bio-based routes meet the "natural" standards defined by the EU and FDA, enabling products to secure a price premium in the market. ( 2 – 5 ) Beyond its role as an endogenous trace compound imparting elegant smoky notes to wine, ( 5 ) 4-ethylphenol (4-EP) holds GRAS (Generally Recognized as Safe) status, solidifying its position as a staple flavoring in smoked foods and seasonings. ( 6 ) Furthermore, the dual functional groups (phenolic hydroxyl and ethyl) on 4-EP make it a versatile key precursor for synthesizing value-added products, including antioxidants, poly(arylene ether) resins, pharmaceuticals, and agrochemicals. ( 7 – 8 ) This broad utility across the food, materials, and pharmaceutical sectors underpins its growing market demand. The prevailing industrial synthesis of 4-EP, based on the petrochemical "sulfonation-alkali fusion-acid precipitation" of ethylbenzene, is fraught with drawbacks. These include high energy consumption for separating closely-boiling ortho-/meta-isomers, complete reliance on non-renewable feedstocks, and severe safety and environmental issues associated with the use of concentrated acids and molten alkalis at high temperatures. ( 9 ) Transitioning to renewable resources is therefore imperative for sustainable production. While catalytic lignin pyrolysis offers an alternative, its practicality is limited by costly catalysts, harsh conditions, and poor efficiency. ( 10 ) A pioneering de novo biosynthetic approach in E. coli (engineered with the TAL-PAD-VPR pathway) demonstrated promise, yet the low solubility and stability of the key vinylphenol reductase (VPR) capped the yield at a modest 110 mg/L. ( 11 ) Consequently, a truly efficient and sustainable strategy for 4-EP production remains an unmet challenge, underscoring the critical need for innovative solutions. Early research into the microbial conversion of p -coumaric acid ( p -CA) to 4-EP proposed two mechanistic routes: a PDC-VPR pathway (decarboxylation first) and a PAR-DEC pathway (reduction first). ( 12 ) While the PAR-DEC route is conceptually attractive for green synthesis, the key enzyme responsible for the decarboxylation step has never been identified in nature, despite the common occurrence of VPR. This absence can be rationalized by the inherent chemical difficulty of cleaving the unactivated C–C bond in the arylpropionic acid side chain—a reaction associated with a high energy barrier even in enzymatic systems. ( 13 ) The development of a robust enzyme catalyst capable of overcoming this barrier to decarboxylate unactivated aromatic acids is therefore a critical unmet goal. Success would not only unlock a novel route to 4-EP but also provide a generalizable "enzyme tool" to facilitate challenging decarboxylations, paving the way for the sustainable production of a wider range of valuable aromatic compounds. Fatty acid photodecarboxylase (FAP), a light-driven enzyme discovered recently, catalyzes the decarboxylation of long-chain fatty acids with exceptional efficiency (turnover numbers up to 8,000) without needing exogenous cofactors. ( 14 – 15 ) Its utility has been greatly expanded beyond natural substrates via directed evolution, which has broadened its scope to include medium- and short-chain acids (C₄–C₁₂), sterically hindered arylalkanoic acids, and epoxidized fatty acids. ( 16 – 18 ) Furthermore, FAP has been demonstrated to enable selective decarboxylation, achieving the kinetic resolution of racemic carboxylic acids, racemic amino acids, and cis/trans isomeric carboxylic acids. ( 19 – 21 ) Leveraging its unique radical mechanism, diverse reactions such as deuterium incorporation, enantioselective decarboxylative cyclization, and dehalogenation have been accomplished. This continuous expansion of its substrate portfolio is establishing FAP as a versatile "photocatalytic interface" for multi-enzyme cascades. Previous work has already realized the conversion of fats to long-chain alkanes and aliphatic epoxides, and of unsaturated fatty acids to stereoselective secondary alcohols, fatty amines, and even the de novo synthesis of ethylbenzene. ( 18 , 25 – 28 ) Its high efficiency and notable substrate promiscuity led us to hypothesize that CvFAP could serve as the sought-after general decarboxylase mentioned above. However, applications of CvFAP to bulky substrates like aromatic carboxylic acids remain scarce. Thus, strategies to enhance CvFAP's compatibility with such substrates through directed evolution, and its effective integration into multi-enzyme cascades, require further in-depth investigation. In this study, we established a novel three-step non-natural pathway, TAL-ER-FAP, for the production of 4-EP from glucose, driven by blue light. This approach achieved a final titer of 4.02 mM, representing the highest yield reported to date. Our work involved several key engineering steps: First, a triple mutant of CvFAP (VVM) with a 3.7-fold increase in catalytic efficiency was obtained through three rounds of saturation mutagenesis. Second, a high-yield tyrosine chassis was constructed based on a previous strategy ( 29 ), achieving a shake-flask titer of 8.6 g/L to supply ample precursor. Third, we co-expressed FjTAL, CaER, and the NADH regeneration enzyme IPADH(M3) ( 30 – 32 ) and employed promoter engineering to balance their protein expression levels ( 33 – 34 ). Ultimately, through a coupled fermentation-biotransformation process, we achieved the synthesis of 4-EP at 4.02 mM. Materials and Methods 2.1 Plasmid and Strain Construction All primers, strains, and plasmids used in this study are listed in Supplementary Tables S1-S7. Phanta max high-fidelity DNA polymerase was used for amplifying all of the targeted genes. The FjTAL, CaER, IPADH(M3), CvFAP were stored by the laboratory, ( 28 ) the aro G fbr and tyr A were amplified from NST-74, ( 35 ) and tktA, ppsA were amplified from W3110(DE3) genome. The purified DNA fragments of FjTAL, CaER, IPADH(M3), aro G fbr , tyr A, tktA, ppsA were cloned into the recipient vectors of pACYCDuet, pRSFDuet, pACYCDuet Str or pTargetF by the T5 Exonuclease-mediated homologous recombination, followed by DNA sequencing verification, resulting in plasmids pRSF-CvFAP, pRSF-CaER-FjTAL, pACYC-IPADH(M3), pACYC str - aro G fbr - tyr A, pTarget-pheA, pTarget-tyrR, pTarget-trpE::tktA, pTarget-pykF::ppsA. Then the pCas9/pTargetF system was employed for genome editing in W3110 (DE3). ( 36 ) 2.2 Medium Luria-Bertani (LB) medium was:10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl. Terrific (TB) Broth medium was:12 g/L tryptone, 24 g/L yeast extract, 0.4% glycerol, 12.5 g/L K 2 HPO 4 , 2.31 g/L KH 2 PO 4 .The fermentation medium for production of ʟ-Tyrosine in shaking flask was: glucose 20 g/L, (NH 4 ) 2 SO 4 10 g/L, KH 2 PO 4 5 g/L, MgSO 4 5 g/L, yeast extract 5 g/L, Tryptone 2g/L, FeSO 4 15 mg/L, sodium citrate 1.5 g/L, thiamine HCl 0.1 g/L and 1mL /L trace metal solution. The pH was adjusted to 6.8 with NH 4 OH. The composition of trace metal solution (per liter): 0.1 M HCl solution, 8.3 g FeCl 3 , 0.84 g ZnCl 2 , 0.13 g CuCl 2 ·2H,0, 0.1 g CoCl 2 ·2H 2 O, 0.1 g H 3 BO 3 , 0.1 g Na 2 MoO 4 and 0.016 g MnCl 2 . 2.3 Cell culture and protein expression The recombinant strain was first inoculated in 1 mL LB medium (containing 50 µg/mL kanamycin or 34 µg/mL chloramphenicol), and the mixture was shaken at 37°C and 200 rpm overnight as preculture. The preculture (2 mL) was transferred into a large culture (50 mL TB + 50 µg/mL kanamycin or 34 µg/mL chloramphenicol in 250-mL shake flasks) at 37°C for about 3–4 h until OD600 reached 0.7.After cooling at 4°C for 15 min, 0.5 mM isopropyl β-thiogalactopyranoside (IPTG) was added to induce the expression of CvFAP and CaER-FjTAL. The culture was further incubated at 22°C, 200 rpm for 12 h. Then, the cells were harvested by centrifugation at 3000× g for 20 min and the supernatants were discarded. The cells were resuspended in 5 mL KP buffer (potassium phosphate buffer, 200 mM, pH 8.0 for CaER-FjTAL, pH 6.5 for CvFAP) and were washed with the same buffer. Finally, the cell suspension was stored at 4 ℃ and used as whole cell catalysts, and protein expression in whole cells was detected using SDS-PAGE (Figure S1). 2.4 Bottle fermentation The strain was first inoculated in 2 mL LB medium (containing 25 µg/mL streptomycin and 17 µg/mL chloramphenicol), and the mixture was shaken at 37°C 200 rpm overnight as preculture. The preculture (2 mL) was transferred into fermentation medium at 37°C for about 2 h until OD600 reached 1.0, isopropyl β-thiogalactopyranoside (IPTG) was added to induce the expression of aro G fbr and tyr A fbr , the culture was further cultured at 30°C, 200 rpm, the pH was maintained at ~ 6.8 with NH 4 OH, and 400 g/L glycerol was used for feeding. 2.5 L-Tyrosine, p -Coumaric acid and p -Hydroxybenzene propanoic acid Quantification by HPLC An SB-C18 column (4.6×250 mm, 5 µm; Agilent Technologies Inc. ) was used to analyze the samples by HPLC. The L-Tyrosine injection volume was 10 µL, the mobile phase was Methanol and 0.1% TFA (30:70, v/v). Benzyl alcohol was used as an internal standard. The p -Coumaric acid and p -Hydroxybenzene propanoic acid injection volume was 6 µL, the mobile phase was Methanol and 0.1% TFA (40:60, v/v), Benzyl acetone was used as an internal reference. The analysis of cascade reactions injection volume was 10 µL, the mobile phase was Methanol and 0.1% TFA (35:65, v/v), Benzyl alcohol was used as an internal standard. The detection wavelength of the UV detector was 210 nm, the flow rate was 1.0 mL/min, the column temperature was 30°C. The relevant reference curves and peak diagrams were respectively at Figure S2-5. 2.6 4-Ethylphenol Quantification by GC MTBE (1 mL) was used for extraction, and GC was used for detection. The conditions for analysis were as follows: 120°C for 1 minutes, increase to 160 ℃ by 20 ℃/min, then hold it for 0.2 minute, increase to 300 ℃ by 80 ℃/min, then hold it for 1 minute. The relevant reference curves and peak diagrams were respectively at Figure S6-7. 2.7 Library construction and site-directed mutagenesis The mutant library was constructed using the 22-codon trick: three PCRs were performed for each site, which were using NDT, VHG, and TGG codons. ( 40 ) Next, a mixture of T4 polynucleotide kinase (Vazyme), T4 DNA ligase (Vazyme), and Dpn I (Lablead) was used to treat the PCR product overnight (25°C). Then the product was transformed into E. coli BL21 (DE3) to obtain the mutant plasmid or plasmid library with the site- specific mutation. The transformation mixture was incubated in 1 mL LB medium at 37 ℃ with shaking, and then spread on LB agar plates containing kanamycin (50 µg/mL). The resulting plasmid or plasmid library was sequenced by Genecreate (Wuhan, China). 2.8 General procedure for blue light reaction The reaction system was 1 mL in a 2-mL vial, containing the following components: the whole cell catalyst (OD600 25), 100 µL HPPA (100 mM, dissolved in KP buffer), and the rest was KP buffer. Using a simple and convenient blue light strip, a blue light reactor was built in the incubating shaker (Figure S8). Next, the reaction bottle was placed in the blue light reactor at16°C. After the reaction was completed, 1 mL methyl tert-butyl ether (MTBE) was added to terminate the reaction. 2.9 Molecular docking Molecular docking simulations are performed by the AutoDock4.2.6 software. ( 37 ) Protein 5NCC is used as the initial template for docking. 4-hydroxybenzenepropanoic is set as the ligand. To include all possible binding conformations, a large cubic box comprised of 50X50X50 girds is used for the docking simulations. Lamarckian genetic algorithm is applied, and each docking calculation contained 10 genetic algorithm runs. ( 38 ) The default values are used for all the other parameters. The first conformation with the highest score is selected as the object of subsequent analysis. 2.10 Molecular dynamics MD simulations were performed using the YASARA. ( 39 ) The AMBER14 force field is applied for the CvFAP protein. After the molecular docking, the conformation with the highest score is selected as the starting point for the subsequent molecular dynamic simulation. The simulations water box adopts the TIP3P water model. Using the specific method of (PME) to handle the long-range Coulomb interactions between charged particles in the simulation. The pH 6.5, using the YASARA defaults for the rest of the parameters. The shape of the simulation cell is Cube, the simulation speed is normal (2*2.25 fs timestep). The duration = 10000 to simulate for 100000 picoseconds. And keeping the solute from diffusing around and crossing periodic boundaries. Using the trajectory analysis module provided by YASARA to analyze the trajectory files. First, the RMSD and RMSF of CvFAP are analyzed. Per-residue contacts with ligand of CvFAP (hydrogen bonds, hydrophobic contacts and ionic interactions) are analyzed by using YASARA md_analysis. mcr. At the same time, the movement of the substrate in the pocket is analyzed by using LigRMSD showing the RMSD of the ligand heavy atoms over time. This procedure delivers information about the movement of the ligand in its binding pocket. Results and Discussion 3.1 Structure-Guided Semi-Rational Engineering of CvFAP Initially, the wild-type CvFAP was confirmed to possess p-hydroxyphenyl propionic acid (HPPA) decarboxylase activity, producing 0.98 mM 4-EP from 10 mM HPPA. However, this activity remained suboptimal compared to reported benchmarks, necessitating further molecular engineering. Structural analysis revealed that the side chain of Y466 protrudes into the substrate channel, narrowing the aperture and potentially hindering the entry of bulky aromatic acids. Substituting this residue with smaller ones yielded variants Y466F and Y466V, which showed 54% and 30% higher conversion, respectively (Fig. 1 A). G462 and P460 are located at the exit of the same hydrophobic pocket, and it was previously confirmed that G462A/P460A could enhance activity towards short-chain fatty acids. ( 16 ) Our prior work also found that introducing the G462I/P460A double mutation increased the conversion of phenylpropionic acid by 95% for the Y466T variant. ( 28 ) In this study, using Y466F and Y466V as templates, we performed a simplified codon-based saturation mutagenesis (22c-trick) at the G462/P460 double sites (Fig. 1 B). This screening identified the superior combinations Y466F/G462V and Y466V/G462V, which exhibited 80% and 88% higher conversion than the wild-type (WT), respectively (Fig. 1 D). Subsequently, single-site saturation mutagenesis at P460 on these bases did not yield further improvement, suggesting that P460 was near its local optimum. Systematic combination of different Y466 mutants with G462A/P460A revealed that Y466F, Y466V, and Y466T all conferred additional activity gains over their parents. Among them, the triple mutant Y466T/G462A/P460A showed the highest activity, a 1.4-fold increase over WT (Fig. 1 D). Notably, the introduction of G462V or G462A/P460A elevated the previously suboptimal Y466V and Y466T templates to top-performing combinations, indicating significant synergistic, rather than simply additive, effects among residues within the pocket. ( 41 ) To further enhance the catalytic efficiency of CvFAP towards HPPA, we performed molecular docking of HPPA into the active site using the PDB ID 5NCC (CvFAP WT) as a template. Thirteen residues within 5 Å of the substrate were selected for saturation mutagenesis (I130, F134, A171, L386, T430, G462, V463, T465, Q486, S573, S574, S575, N620; green region in Fig. 1 C), using Y466T/G462A/P460A and Y466V/G462V as templates. However, no variants with improved activity were obtained. Subsequently, we focused on the substrate tunnel by targeting six residues within 4 Å of the native fatty acid substrate (V453, G455, M456, A457, F469, T484; blue region in Fig. 1 C). In this round of screening, position V453 stood out. The V453L and V453M mutations increased the conversion rate by 50% and 151%, respectively. The optimal triple mutant (Y466V/G462V/V453M) achieved a product titer of 4.6 mM, representing a 4.7-fold increase over the wild-type. Structural analysis revealed that Y466, V453, and A457 collectively form the narrowest constriction within the CvFAP channel (Fig. 1 E), which may explain the significant activity enhancement conferred by the V453 mutation. It is noteworthy that V453E was previously reported to differentiate trans-fatty acids. ( 21 ) Our finding that beneficial mutations at V453 were identified in two independent templates suggests that, like Y466 and G462, V453 has the potential to be a key hotspot for engineering CvFAP. Prioritizing these residues could enable rapid enhancement of CvFAP activity. 3.2 Molecular Dynamics (MD) Simulations To elucidate the molecular basis for the enhanced activity of the VVM mutant, we constructed a homology model of CvFAP (Y466V/G462V/V453M). Molecular docking (Figure S3) and 100-ns molecular dynamics (MD) simulations were then performed based on this model and the wild-type (WT) crystal structure. The distance between the substrate's carboxyl group and the flavin moiety of the FAD cofactor is critical for catalytic activity, as a shorter distance facilitates electron transfer in the initial catalytic step. ( 21 ) The MD simulations revealed a significant change in this distance. The average distance between the substrate's carboxyl oxygen and the N5 atom of FAD decreased from 7.758 Å in the WT to 5.457 Å in the VVM mutant (Fig. 2 B). Furthermore, the substrate exhibited a lower root-mean-square deviation (RMSD) within the binding pocket of the mutant (Fig. 2 C), indicating more precise positioning of the carboxyl group and reduced overall fluctuation, thereby contributing to higher catalytic efficiency. Additionally, the conserved residue R451 in the VVM mutant formed a stronger salt bridge interaction with the substrate. Concurrently, the G462V mutation enhanced hydrophobic interactions at this position, collectively pushing the substrate closer to the FAD cofactor and improving catalytic efficiency (Fig. 2 D). 3.3 Construction of a High-Yield L-Tyrosine Chassis To achieve the synthesis of 4-EP from renewable sugar sources, the priority is to construct an efficient tyrosine-producing chassis. Strategies such as knocking out Phe A and Tyr R to reduce pathway competition and feedback inhibition, or overexpressing genes like aro G, tyr A, tkt A, and pps A to enhance precursor supply and direct tyrosine synthesis, have been widely used in constructing tyrosine-producing strains. Ping et al. systematically evaluated 16 related genes and confirmed that the knockout combination tyr R/pheA/ trp E yielded the highest titer, reaching 3.76 g/L in shake flasks at 48 hours. Conversely, the overexpression combination aro G fbr / tyr A fbr / tkt A/ pps A also achieved a high titer of 1.84 g/L at 48 hours. The strain HGA1, obtained by combining these two strategies, showed an increased tyrosine production of 4.22 g/L.( 28 ) To efficiently obtain the tyrosine precursor, we first knocked out phe A and tyr R to block the phenylalanine branch and relieve feedback inhibition, obtaining a base strain. Subsequently, we overexpressed aro Gfbr and tyr Afbr in this background to construct Strain 1. Building upon this, we further knocked out trp E and integrated tkt A in situ (Strain 2), and then replaced pyk F with pps A (Strain 3) to enhance the supply of phosphoenolpyruvate and erythrose-4-phosphate. Without IPTG induction, Strain 3 accumulated 1.61 g/L of tyrosine at 24 hours, a 48% increase compared to Strain 1. After optimizing the IPTG concentration gradient, the highest yield reached 4.23 g/L at 72 hours under 40 µM IPTG. In a fed-batch shake flask system with a total sugar concentration of 40 g/L, this strain produced 8.6 g/L of tyrosine, with a substrate conversion rate of 0.215 g/g. The resulting fermentation broth can be directly used for the subsequent photo-enzymatic cascaded synthesis of 4-EP without requiring additional purification. 3.3 Multi-Enzyme Cascade Conversion of L-Tyrosine to 4-EP The reduction of the double bond from p -coumaric acid to p -hydroxyphenyl propionic acid (HPPA) requires a suitable catalyst. In biocatalysis, Old Yellow Enzymes (OYEs) and enoate reductases (ERs) are typically employed for such reactions. OYEs are primarily used for the asymmetric reduction of activated alkenes, while ERs have been less characterized due to their oxygen sensitivity. ( 42 ) Previously, Sun et al. identified an ER from Clostridium acetobutylicum (CaER) that remained active under aerobic conditions and pioneered the de novo synthesis of HPPA and 3PPA, ( 31 ) providing a feasibility basis for this study. To reduce process costs, we employed a whole-cell cascade catalysis system for HPPA synthesis: FjTAL and CaER were co-expressed on a pRSF-Duet vector, and IPADH(M3) was introduced to regenerate NADH. ( 32 )Through orthogonal optimization of pH and temperature, we found that HPPA production was highest at 30°C and pH 8.0. While higher temperatures or pH values increased FjTAL activity, they inhibited CaER, and vice versa. Modulating promoter or RBS strength is a common strategy in multi-enzyme cascades to balance protein expression levels. ( 33 ) Here, we used four T7 promoter mutants of varying strengths (TA, TT, AA, AT, from strong to weak) in pairwise combinations to fine-tune the expression ratio of FjTAL to CaER. ( 34 ) The results showed that the medium-strength AA-AA combination yielded the highest HPPA titer of 26.7 mM, providing ample substrate for the subsequent photodecarboxylation step. After determining the catalytic capacity of each cell catalyst, we used 2 g/L (DCW) of the TAL-ER strain and 10 g/L (DCW) of the FAP strain to convert 5 mM tyrosine from the fermentation broth. Due to the significantly different optimal conditions for the two catalysts, we adopted a sequential reaction strategy. ( 43 ) First, 2 g/L (DCW) of the TAL-ER cells completely converted 5 mM tyrosine to p-coumaric acid within 8 hours at 30°C and pH 8.0. Subsequently, 10 g/L (DCW) of the FAP cells were added, and the conditions were adjusted to 16°C and pH 6.5 for a further 2-hour reaction. This process achieved a final 4-EP concentration of 4.02 mM, which is the highest level reported to date. In summary, this study leverages the innate substrate promiscuity of fatty acid photodecarboxylase (FAP). Through directed evolution, we enhanced its catalytic efficiency for a non-native aromatic substrate by 3.7-fold and identified Y466, G462, P460, and V453 as key engineering hotspots, providing a general strategy for broadening FAP's substrate scope. To achieve de novo synthesis of 4-EP, we constructed a high-yield L-tyrosine chassis (8.6 g/L in shake flasks) following Ping et al., which served as the foundation for the multi-enzyme cascade. Promoter engineering optimized the pathway, elevating the HPPA titer to 26.7 mM. We identified the mismatched pH optima of FjTAL and CaER, along with the oxygen sensitivity of CaER, as the primary bottlenecks for the efficient conversion of L-Tyr to HPPA; this conversion could potentially be further improved by employing acid-tolerant TALs and anaerobic catalysis. Finally, the optimized FAP mutant quantitatively decarboxylated HPPA, yielding a final 4-EP concentration of 4.02 mM—the highest value reported to date. Our work demonstrates that FAP, with its high efficiency and broad promiscuity, can be integrated as a versatile "decarboxylation platform module" into biosynthetic pathways to overcome challenging decarboxylation steps, offering a new paradigm for the green synthesis of aromatic chemicals. Declarations Competing interests The authors declare no competing interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 32100060, 32101229), Fundamental Research Funds for the Central Universities (No. 2662021SKQD001, 2662021JC006), and the LongYun Program 2.0 from College of Life Science and Technology. References Gosset, G. Production of Aromatic Compounds in Bacteria. Current Opinion in Biotechnology 2009 , 20 (6), 651–658. https://doi.org/10.1016/j.copbio.2009.09.012. Thompson, B.; Machas, M.; Nielsen, D. R. Creating Pathways towards Aromatic Building Blocks and Fine Chemicals. Current Opinion in Biotechnology 2015 , 36 , 1–7. https://doi.org/10.1016/j.copbio.2015.07.004. Shen, Y.-P.; Niu, F.-X.; Yan, Z.-B.; Fong, L. S.; Huang, Y.-B.; Liu, J.-Z. Recent Advances in Metabolically Engineered Microorganisms for the Production of Aromatic Chemicals Derived From Aromatic Amino Acids. Front. Bioeng. Biotechnol. 2020 , 8 , 407. https://doi.org/10.3389/fbioe.2020.00407. Paulino, B. 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09:33:59","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131398,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/dc9fd7e16f718b83e219851b.html"},{"id":92398215,"identity":"ec9fc7ea-31fb-4839-a289-55e04d9fab8e","added_by":"auto","created_at":"2025-09-29 09:41:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341471,"visible":true,"origin":"","legend":"\u003cp\u003eEngineering of CvFAP (A) Decarboxylation of HPPA to produce 4-EP with CvFAP variants substituted at position Y466. (B) Saturation Mutagenesis at G462 and P460 on the Y466F and Y466V Templates.(C) Model of the substrate entrance tunnel of CvFAP (PDB ID: 5NCC) shown as a gray surface representation. The coenzyme FAD is represented by rainbow sticks, and HPPA (by docking) is depicted as rainbow spheres. The green and blue colors represent residues within 5 Å of HPPA and 4 Å of the native substrate, respectively.(D) Different CvFAP variants obtained by directed evolution.(E) The minimum cross-section of the CvFAP tunnel, lined by residues Y466, A457, and V453.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/4e083055b57b876110978248.png"},{"id":92396827,"identity":"a38dc159-0a7d-4822-bcc6-8d31588306c9","added_by":"auto","created_at":"2025-09-29 09:33:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":454506,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular Dynamics Simulation of CvFAP (A) The distance between the FAD-N5 atom and the HPPA-O1 is indicated by a dashed line. (B) The Distance between FAD-N5 atom and the HPPA-O1 atom of CvFAP(WT) and CvFAP(VVM) during 100 ns MD simulation.(C) The RMSD of HPPA in the two variants during 100 ns MD simulation.(D). Analysis of the interaction between the substrate and CvFAP.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/d3edd4a1d4f886cd504c935e.png"},{"id":92396825,"identity":"99f436d2-782b-46c6-a037-ac85f32bab36","added_by":"auto","created_at":"2025-09-29 09:33:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":163181,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of a Tyrosine Fermentation Strain (A) Schematic diagram of the design for the L-tyrosine fermentation strain. Red annotations indicate overexpressed genes and crossed-out arrows indicate knocked-out genes. (B) Results of 24-hour shaker fermentation for different strains. (C) Results of 72-hour shaker fermentation under IPTG optimization.(D) Batch-fed fermentation with glucose at a total concentration of 40 g/L.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/f30747beadeb2871295120f2.png"},{"id":92398617,"identity":"b5c3c84c-c25e-498c-b3ff-a684b1dbd8a9","added_by":"auto","created_at":"2025-09-29 09:49:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":254395,"visible":true,"origin":"","legend":"\u003cp\u003eMulti-enzyme Cascade Conversion of Tyrosine to 4-EP (A) Orthogonal Optimization of pH and Temperature for the Conversion of L-Tyr to HPPA. (B) Balancing FjTAL and CaER expression using a panel of four T7 promoter mutants of varying strengths. Preliminary screening was performed in 24-well plates. (C) Precise evaluation of eight selected combinations from Figure 4B using shake-flask cultivation..(D). Time course of the cascade biotransformation of L-Tyr (fermentation broth) into 4-EP with two different E. coli cultures in sequential mode. The reaction was performed at 30 °C for 8 h before turning on the blue light and adjusting the shaker temperature to 16 °C.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/911e1418520f27128f071218.png"},{"id":92400020,"identity":"abdd7910-b134-4bca-9b12-8fa6a8022930","added_by":"auto","created_at":"2025-09-29 10:05:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1898952,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/0f13e356-2798-45ac-ac58-ff3e1bbb960e.pdf"},{"id":92396824,"identity":"0d90196f-8a8e-4b7c-a725-dff1a924dbdc","added_by":"auto","created_at":"2025-09-29 09:33:58","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":55563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1. \u003c/strong\u003eA non-natural enzyme cascade to produce 4-ethylbenzene from biobased L-Tyrosine, which can be obtained by \u003cem\u003eE. coli\u003c/em\u003ecultivation.\u003c/p\u003e","description":"","filename":"SCHEME1.png","url":"https://assets-eu.researchsquare.com/files/rs-7714953/v1/824b7f02d1e8615356286c9d.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eBiosynthesis of 4-ethylphenol via an enzyme cascade with an engineered fatty acid photodecarboxylase\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAromatic chemicals are pivotal \"functional cores\" in diverse sectors\u0026mdash;including pharmaceuticals, flavors, cosmetics, food, and advanced materials\u0026mdash;owing to their distinct chemical properties. Conventional production, however, depends on petrochemical feedstocks, which is fraught with drawbacks: resource depletion, price instability, high energy costs, and substantial environmental footprint. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Shifting to renewable resources for sustainable synthesis is thus a vital strategic pivot. This transition is being accelerated by synthetic biology and enzyme engineering, which have elevated the microbial production of aromatics from laboratory-scale demonstrations to commercial reality. Key compounds such as vanillin, cinnamic acid, and coumarin are now produced through whole-cell biocatalysis or multi-enzyme systems; crucially, these bio-based routes meet the \"natural\" standards defined by the EU and FDA, enabling products to secure a price premium in the market. (\u003cspan additionalcitationids=\"CR3 CR4\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eBeyond its role as an endogenous trace compound imparting elegant smoky notes to wine, (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) 4-ethylphenol (4-EP) holds GRAS (Generally Recognized as Safe) status, solidifying its position as a staple flavoring in smoked foods and seasonings. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Furthermore, the dual functional groups (phenolic hydroxyl and ethyl) on 4-EP make it a versatile key precursor for synthesizing value-added products, including antioxidants, poly(arylene ether) resins, pharmaceuticals, and agrochemicals. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) This broad utility across the food, materials, and pharmaceutical sectors underpins its growing market demand.\u003c/p\u003e\u003cp\u003eThe prevailing industrial synthesis of 4-EP, based on the petrochemical \"sulfonation-alkali fusion-acid precipitation\" of ethylbenzene, is fraught with drawbacks. These include high energy consumption for separating closely-boiling ortho-/meta-isomers, complete reliance on non-renewable feedstocks, and severe safety and environmental issues associated with the use of concentrated acids and molten alkalis at high temperatures. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) Transitioning to renewable resources is therefore imperative for sustainable production. While catalytic lignin pyrolysis offers an alternative, its practicality is limited by costly catalysts, harsh conditions, and poor efficiency. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) A pioneering de novo biosynthetic approach in E. coli (engineered with the TAL-PAD-VPR pathway) demonstrated promise, yet the low solubility and stability of the key vinylphenol reductase (VPR) capped the yield at a modest 110 mg/L. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) Consequently, a truly efficient and sustainable strategy for 4-EP production remains an unmet challenge, underscoring the critical need for innovative solutions.\u003c/p\u003e\u003cp\u003eEarly research into the microbial conversion of \u003cem\u003ep\u003c/em\u003e-coumaric acid (\u003cem\u003ep\u003c/em\u003e-CA) to 4-EP proposed two mechanistic routes: a PDC-VPR pathway (decarboxylation first) and a PAR-DEC pathway (reduction first). (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) While the PAR-DEC route is conceptually attractive for green synthesis, the key enzyme responsible for the decarboxylation step has never been identified in nature, despite the common occurrence of VPR. This absence can be rationalized by the inherent chemical difficulty of cleaving the unactivated C\u0026ndash;C bond in the arylpropionic acid side chain\u0026mdash;a reaction associated with a high energy barrier even in enzymatic systems. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e) The development of a robust enzyme catalyst capable of overcoming this barrier to decarboxylate unactivated aromatic acids is therefore a critical unmet goal. Success would not only unlock a novel route to 4-EP but also provide a generalizable \"enzyme tool\" to facilitate challenging decarboxylations, paving the way for the sustainable production of a wider range of valuable aromatic compounds.\u003c/p\u003e\u003cp\u003eFatty acid photodecarboxylase (FAP), a light-driven enzyme discovered recently, catalyzes the decarboxylation of long-chain fatty acids with exceptional efficiency (turnover numbers up to 8,000) without needing exogenous cofactors. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) Its utility has been greatly expanded beyond natural substrates via directed evolution, which has broadened its scope to include medium- and short-chain acids (C₄\u0026ndash;C₁₂), sterically hindered arylalkanoic acids, and epoxidized fatty acids. (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e) Furthermore, FAP has been demonstrated to enable selective decarboxylation, achieving the kinetic resolution of racemic carboxylic acids, racemic amino acids, and cis/trans isomeric carboxylic acids. (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) Leveraging its unique radical mechanism, diverse reactions such as deuterium incorporation, enantioselective decarboxylative cyclization, and dehalogenation have been accomplished. This continuous expansion of its substrate portfolio is establishing FAP as a versatile \"photocatalytic interface\" for multi-enzyme cascades. Previous work has already realized the conversion of fats to long-chain alkanes and aliphatic epoxides, and of unsaturated fatty acids to stereoselective secondary alcohols, fatty amines, and even the de novo synthesis of ethylbenzene. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) Its high efficiency and notable substrate promiscuity led us to hypothesize that CvFAP could serve as the sought-after general decarboxylase mentioned above. However, applications of CvFAP to bulky substrates like aromatic carboxylic acids remain scarce. Thus, strategies to enhance CvFAP's compatibility with such substrates through directed evolution, and its effective integration into multi-enzyme cascades, require further in-depth investigation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study, we established a novel three-step non-natural pathway, TAL-ER-FAP, for the production of 4-EP from glucose, driven by blue light. This approach achieved a final titer of 4.02 mM, representing the highest yield reported to date. Our work involved several key engineering steps: First, a triple mutant of CvFAP (VVM) with a 3.7-fold increase in catalytic efficiency was obtained through three rounds of saturation mutagenesis. Second, a high-yield tyrosine chassis was constructed based on a previous strategy (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), achieving a shake-flask titer of 8.6 g/L to supply ample precursor. Third, we co-expressed FjTAL, CaER, and the NADH regeneration enzyme IPADH(M3) (\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) and employed promoter engineering to balance their protein expression levels (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Ultimately, through a coupled fermentation-biotransformation process, we achieved the synthesis of 4-EP at 4.02 mM.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Plasmid and Strain Construction\u003c/h2\u003e\u003cp\u003eAll primers, strains, and plasmids used in this study are listed in Supplementary Tables S1-S7. Phanta max high-fidelity DNA polymerase was used for amplifying all of the targeted genes. The FjTAL, CaER, IPADH(M3), CvFAP were stored by the laboratory, (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) the \u003cem\u003earo\u003c/em\u003eG\u003csup\u003efbr\u003c/sup\u003e and \u003cem\u003etyr\u003c/em\u003eA were amplified from NST-74, (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e) and tktA, ppsA were amplified from W3110(DE3) genome.\u003c/p\u003e\u003cp\u003eThe purified DNA fragments of FjTAL, CaER, IPADH(M3), \u003cem\u003earo\u003c/em\u003eG\u003csup\u003efbr\u003c/sup\u003e, \u003cem\u003etyr\u003c/em\u003eA, tktA, ppsA were cloned into the recipient vectors of pACYCDuet, pRSFDuet, pACYCDuet\u003csup\u003eStr\u003c/sup\u003e or pTargetF by the T5 Exonuclease-mediated homologous recombination, followed by DNA sequencing verification, resulting in plasmids pRSF-CvFAP, pRSF-CaER-FjTAL, pACYC-IPADH(M3), pACYC\u003csup\u003estr\u003c/sup\u003e-\u003cem\u003earo\u003c/em\u003eG\u003csup\u003efbr\u003c/sup\u003e-\u003cem\u003etyr\u003c/em\u003eA, pTarget-pheA, pTarget-tyrR, pTarget-trpE::tktA, pTarget-pykF::ppsA. Then the pCas9/pTargetF system was employed for genome editing in W3110 (DE3). (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Medium\u003c/h2\u003e\u003cp\u003eLuria-Bertani (LB) medium was:10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl. Terrific (TB) Broth medium was:12 g/L tryptone, 24 g/L yeast extract, 0.4% glycerol, 12.5 g/L K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2.31 g/L KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e.The fermentation medium for production of ʟ-Tyrosine in shaking flask was: glucose 20 g/L, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e 10 g/L, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 5 g/L, MgSO\u003csub\u003e4\u003c/sub\u003e 5 g/L, yeast extract 5 g/L, Tryptone 2g/L, FeSO\u003csub\u003e4\u003c/sub\u003e 15 mg/L, sodium citrate 1.5 g/L, thiamine HCl 0.1 g/L and 1mL /L trace metal solution. The pH was adjusted to 6.8 with NH\u003csub\u003e4\u003c/sub\u003eOH. The composition of trace metal solution (per liter): 0.1 M HCl solution, 8.3 g FeCl\u003csub\u003e3\u003c/sub\u003e, 0.84 g ZnCl\u003csub\u003e2\u003c/sub\u003e, 0.13 g CuCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H,0, 0.1 g CoCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO, 0.1 g H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, 0.1 g Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e and 0.016 g MnCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell culture and protein expression\u003c/h2\u003e\u003cp\u003eThe recombinant strain was first inoculated in 1 mL LB medium (containing 50 \u0026micro;g/mL kanamycin or 34 \u0026micro;g/mL chloramphenicol), and the mixture was shaken at 37\u0026deg;C and 200 rpm overnight as preculture. The preculture (2 mL) was transferred into a large culture (50 mL TB\u0026thinsp;+\u0026thinsp;50 \u0026micro;g/mL kanamycin or 34 \u0026micro;g/mL chloramphenicol in 250-mL shake flasks) at 37\u0026deg;C for about 3\u0026ndash;4 h until OD600 reached 0.7.After cooling at 4\u0026deg;C for 15 min, 0.5 mM isopropyl β-thiogalactopyranoside (IPTG) was added to induce the expression of CvFAP and CaER-FjTAL. The culture was further incubated at 22\u0026deg;C, 200 rpm for 12 h. Then, the cells were harvested by centrifugation at 3000\u0026times; \u003cem\u003eg\u003c/em\u003e for 20 min and the supernatants were discarded. The cells were resuspended in 5 mL KP buffer (potassium phosphate buffer, 200 mM, pH 8.0 for CaER-FjTAL, pH 6.5 for CvFAP) and were washed with the same buffer. Finally, the cell suspension was stored at 4 ℃ and used as whole cell catalysts, and protein expression in whole cells was detected using SDS-PAGE (Figure S1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Bottle fermentation\u003c/h2\u003e\u003cp\u003eThe strain was first inoculated in 2 mL LB medium (containing 25 \u0026micro;g/mL streptomycin and 17 \u0026micro;g/mL chloramphenicol), and the mixture was shaken at 37\u0026deg;C 200 rpm overnight as preculture. The preculture (2 mL) was transferred into fermentation medium at 37\u0026deg;C for about 2 h until OD600 reached 1.0, isopropyl β-thiogalactopyranoside (IPTG) was added to induce the expression of \u003cem\u003earo\u003c/em\u003eG\u003csup\u003efbr\u003c/sup\u003e and \u003cem\u003etyr\u003c/em\u003eA\u003csup\u003efbr\u003c/sup\u003e, the culture was further cultured at 30\u0026deg;C, 200 rpm, the pH was maintained at ~\u0026thinsp;6.8 with NH\u003csub\u003e4\u003c/sub\u003eOH, and 400 g/L glycerol was used for feeding.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 L-Tyrosine, \u003cem\u003ep\u003c/em\u003e-Coumaric acid and \u003cem\u003ep\u003c/em\u003e-Hydroxybenzene propanoic acid Quantification by HPLC\u003c/h2\u003e\u003cp\u003eAn SB-C18 column (4.6\u0026times;250 mm, 5 \u0026micro;m; Agilent Technologies Inc. ) was used to analyze the samples by HPLC. The L-Tyrosine injection volume was 10 \u0026micro;L, the mobile phase was Methanol and 0.1% TFA (30:70, v/v). Benzyl alcohol was used as an internal standard. The \u003cem\u003ep\u003c/em\u003e-Coumaric acid and \u003cem\u003ep\u003c/em\u003e-Hydroxybenzene propanoic acid injection volume was 6 \u0026micro;L, the mobile phase was Methanol and 0.1% TFA (40:60, v/v), Benzyl acetone was used as an internal reference. The analysis of cascade reactions injection volume was 10 \u0026micro;L, the mobile phase was Methanol and 0.1% TFA (35:65, v/v), Benzyl alcohol was used as an internal standard. The detection wavelength of the UV detector was 210 nm, the flow rate was 1.0 mL/min, the column temperature was 30\u0026deg;C. The relevant reference curves and peak diagrams were respectively at Figure S2-5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 4-Ethylphenol Quantification by GC\u003c/h2\u003e\u003cp\u003eMTBE (1 mL) was used for extraction, and GC was used for detection. The conditions for analysis were as follows: 120\u0026deg;C for 1 minutes, increase to 160 ℃ by 20 ℃/min, then hold it for 0.2 minute, increase to 300 ℃ by 80 ℃/min, then hold it for 1 minute. The relevant reference curves and peak diagrams were respectively at Figure S6-7.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Library construction and site-directed mutagenesis\u003c/h2\u003e\u003cp\u003eThe mutant library was constructed using the 22-codon trick: three PCRs were performed for each site, which were using NDT, VHG, and TGG codons. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) Next, a mixture of T4 polynucleotide kinase (Vazyme), T4 DNA ligase (Vazyme), and \u003cem\u003eDpn\u003c/em\u003e I (Lablead) was used to treat the PCR product overnight (25\u0026deg;C). Then the product was transformed into E. coli BL21 (DE3) to obtain the mutant plasmid or plasmid library with the site- specific mutation. The transformation mixture was incubated in 1 mL LB medium at 37 ℃ with shaking, and then spread on LB agar plates containing kanamycin (50 \u0026micro;g/mL). The resulting plasmid or plasmid library was sequenced by Genecreate (Wuhan, China).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 General procedure for blue light reaction\u003c/h2\u003e\u003cp\u003eThe reaction system was 1 mL in a 2-mL vial, containing the following components: the whole cell catalyst (OD600 25), 100 \u0026micro;L HPPA (100 mM, dissolved in KP buffer), and the rest was KP buffer. Using a simple and convenient blue light strip, a blue light reactor was built in the incubating shaker (Figure S8). Next, the reaction bottle was placed in the blue light reactor at16\u0026deg;C. After the reaction was completed, 1 mL methyl tert-butyl ether (MTBE) was added to terminate the reaction.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Molecular docking\u003c/h2\u003e\u003cp\u003eMolecular docking simulations are performed by the AutoDock4.2.6 software. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e) Protein 5NCC is used as the initial template for docking. 4-hydroxybenzenepropanoic is set as the ligand. To include all possible binding conformations, a large cubic box comprised of 50X50X50 girds is used for the docking simulations. Lamarckian genetic algorithm is applied, and each docking calculation contained 10 genetic algorithm runs. (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) The default values are used for all the other parameters. The first conformation with the highest score is selected as the object of subsequent analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Molecular dynamics\u003c/h2\u003e\u003cp\u003eMD simulations were performed using the YASARA. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) The AMBER14 force field is applied for the CvFAP protein. After the molecular docking, the conformation with the highest score is selected as the starting point for the subsequent molecular dynamic simulation. The simulations water box adopts the TIP3P water model. Using the specific method of (PME) to handle the long-range Coulomb interactions between charged particles in the simulation. The pH 6.5, using the YASARA defaults for the rest of the parameters. The shape of the simulation cell is Cube, the simulation speed is normal (2*2.25 fs timestep). The duration\u0026thinsp;=\u0026thinsp;10000 to simulate for 100000 picoseconds. And keeping the solute from diffusing around and crossing periodic boundaries. Using the trajectory analysis module provided by YASARA to analyze the trajectory files. First, the RMSD and RMSF of CvFAP are analyzed. Per-residue contacts with ligand of CvFAP (hydrogen bonds, hydrophobic contacts and ionic interactions) are analyzed by using YASARA md_analysis. mcr. At the same time, the movement of the substrate in the pocket is analyzed by using LigRMSD showing the RMSD of the ligand heavy atoms over time. This procedure delivers information about the movement of the ligand in its binding pocket.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Structure-Guided Semi-Rational Engineering of CvFAP\u003c/h2\u003e\u003cp\u003eInitially, the wild-type CvFAP was confirmed to possess p-hydroxyphenyl propionic acid (HPPA) decarboxylase activity, producing 0.98 mM 4-EP from 10 mM HPPA. However, this activity remained suboptimal compared to reported benchmarks, necessitating further molecular engineering. Structural analysis revealed that the side chain of Y466 protrudes into the substrate channel, narrowing the aperture and potentially hindering the entry of bulky aromatic acids. Substituting this residue with smaller ones yielded variants Y466F and Y466V, which showed 54% and 30% higher conversion, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). G462 and P460 are located at the exit of the same hydrophobic pocket, and it was previously confirmed that G462A/P460A could enhance activity towards short-chain fatty acids. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) Our prior work also found that introducing the G462I/P460A double mutation increased the conversion of phenylpropionic acid by 95% for the Y466T variant. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) In this study, using Y466F and Y466V as templates, we performed a simplified codon-based saturation mutagenesis (22c-trick) at the G462/P460 double sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This screening identified the superior combinations Y466F/G462V and Y466V/G462V, which exhibited 80% and 88% higher conversion than the wild-type (WT), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Subsequently, single-site saturation mutagenesis at P460 on these bases did not yield further improvement, suggesting that P460 was near its local optimum. Systematic combination of different Y466 mutants with G462A/P460A revealed that Y466F, Y466V, and Y466T all conferred additional activity gains over their parents. Among them, the triple mutant Y466T/G462A/P460A showed the highest activity, a 1.4-fold increase over WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Notably, the introduction of G462V or G462A/P460A elevated the previously suboptimal Y466V and Y466T templates to top-performing combinations, indicating significant synergistic, rather than simply additive, effects among residues within the pocket. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eTo further enhance the catalytic efficiency of CvFAP towards HPPA, we performed molecular docking of HPPA into the active site using the PDB ID 5NCC (CvFAP WT) as a template. Thirteen residues within 5 \u0026Aring; of the substrate were selected for saturation mutagenesis (I130, F134, A171, L386, T430, G462, V463, T465, Q486, S573, S574, S575, N620; green region in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), using Y466T/G462A/P460A and Y466V/G462V as templates. However, no variants with improved activity were obtained. Subsequently, we focused on the substrate tunnel by targeting six residues within 4 \u0026Aring; of the native fatty acid substrate (V453, G455, M456, A457, F469, T484; blue region in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In this round of screening, position V453 stood out. The V453L and V453M mutations increased the conversion rate by 50% and 151%, respectively. The optimal triple mutant (Y466V/G462V/V453M) achieved a product titer of 4.6 mM, representing a 4.7-fold increase over the wild-type. Structural analysis revealed that Y466, V453, and A457 collectively form the narrowest constriction within the CvFAP channel (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), which may explain the significant activity enhancement conferred by the V453 mutation. It is noteworthy that V453E was previously reported to differentiate trans-fatty acids. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) Our finding that beneficial mutations at V453 were identified in two independent templates suggests that, like Y466 and G462, V453 has the potential to be a key hotspot for engineering CvFAP. Prioritizing these residues could enable rapid enhancement of CvFAP activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Molecular Dynamics (MD) Simulations\u003c/h2\u003e\u003cp\u003eTo elucidate the molecular basis for the enhanced activity of the VVM mutant, we constructed a homology model of CvFAP (Y466V/G462V/V453M). Molecular docking (Figure S3) and 100-ns molecular dynamics (MD) simulations were then performed based on this model and the wild-type (WT) crystal structure. The distance between the substrate's carboxyl group and the flavin moiety of the FAD cofactor is critical for catalytic activity, as a shorter distance facilitates electron transfer in the initial catalytic step. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) The MD simulations revealed a significant change in this distance. The average distance between the substrate's carboxyl oxygen and the N5 atom of FAD decreased from 7.758 \u0026Aring; in the WT to 5.457 \u0026Aring; in the VVM mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, the substrate exhibited a lower root-mean-square deviation (RMSD) within the binding pocket of the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), indicating more precise positioning of the carboxyl group and reduced overall fluctuation, thereby contributing to higher catalytic efficiency. Additionally, the conserved residue R451 in the VVM mutant formed a stronger salt bridge interaction with the substrate. Concurrently, the G462V mutation enhanced hydrophobic interactions at this position, collectively pushing the substrate closer to the FAD cofactor and improving catalytic efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Construction of a High-Yield L-Tyrosine Chassis\u003c/h2\u003e\u003cp\u003eTo achieve the synthesis of 4-EP from renewable sugar sources, the priority is to construct an efficient tyrosine-producing chassis. Strategies such as knocking out \u003cem\u003ePhe\u003c/em\u003eA and \u003cem\u003eTyr\u003c/em\u003eR to reduce pathway competition and feedback inhibition, or overexpressing genes like \u003cem\u003earo\u003c/em\u003eG, \u003cem\u003etyr\u003c/em\u003eA, \u003cem\u003etkt\u003c/em\u003eA, and \u003cem\u003epps\u003c/em\u003eA to enhance precursor supply and direct tyrosine synthesis, have been widely used in constructing tyrosine-producing strains. Ping et al. systematically evaluated 16 related genes and confirmed that the knockout combination \u003cem\u003etyr\u003c/em\u003eR/pheA/\u003cem\u003etrp\u003c/em\u003eE yielded the highest titer, reaching 3.76 g/L in shake flasks at 48 hours. Conversely, the overexpression combination \u003cem\u003earo\u003c/em\u003eG\u003csup\u003efbr\u003c/sup\u003e/\u003cem\u003etyr\u003c/em\u003eA\u003csup\u003efbr\u003c/sup\u003e/\u003cem\u003etkt\u003c/em\u003eA/\u003cem\u003epps\u003c/em\u003eA also achieved a high titer of 1.84 g/L at 48 hours. The strain HGA1, obtained by combining these two strategies, showed an increased tyrosine production of 4.22 g/L.(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eTo efficiently obtain the tyrosine precursor, we first knocked out \u003cem\u003ephe\u003c/em\u003eA and \u003cem\u003etyr\u003c/em\u003eR to block the phenylalanine branch and relieve feedback inhibition, obtaining a base strain. Subsequently, we overexpressed \u003cem\u003earo\u003c/em\u003eGfbr and \u003cem\u003etyr\u003c/em\u003eAfbr in this background to construct Strain 1. Building upon this, we further knocked out \u003cem\u003etrp\u003c/em\u003eE and integrated \u003cem\u003etkt\u003c/em\u003eA in situ (Strain 2), and then replaced \u003cem\u003epyk\u003c/em\u003eF with \u003cem\u003epps\u003c/em\u003eA (Strain 3) to enhance the supply of phosphoenolpyruvate and erythrose-4-phosphate. Without IPTG induction, Strain 3 accumulated 1.61 g/L of tyrosine at 24 hours, a 48% increase compared to Strain 1. After optimizing the IPTG concentration gradient, the highest yield reached 4.23 g/L at 72 hours under 40 \u0026micro;M IPTG. In a fed-batch shake flask system with a total sugar concentration of 40 g/L, this strain produced 8.6 g/L of tyrosine, with a substrate conversion rate of 0.215 g/g. The resulting fermentation broth can be directly used for the subsequent photo-enzymatic cascaded synthesis of 4-EP without requiring additional purification.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Multi-Enzyme Cascade Conversion of L-Tyrosine to 4-EP\u003c/h2\u003e\u003cp\u003eThe reduction of the double bond from \u003cem\u003ep\u003c/em\u003e-coumaric acid to \u003cem\u003ep\u003c/em\u003e-hydroxyphenyl propionic acid (HPPA) requires a suitable catalyst. In biocatalysis, Old Yellow Enzymes (OYEs) and enoate reductases (ERs) are typically employed for such reactions. OYEs are primarily used for the asymmetric reduction of activated alkenes, while ERs have been less characterized due to their oxygen sensitivity. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) Previously, Sun et al. identified an ER from Clostridium acetobutylicum (CaER) that remained active under aerobic conditions and pioneered the de novo synthesis of HPPA and 3PPA, (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) providing a feasibility basis for this study. To reduce process costs, we employed a whole-cell cascade catalysis system for HPPA synthesis: FjTAL and CaER were co-expressed on a pRSF-Duet vector, and IPADH(M3) was introduced to regenerate NADH. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)Through orthogonal optimization of pH and temperature, we found that HPPA production was highest at 30\u0026deg;C and pH 8.0. While higher temperatures or pH values increased FjTAL activity, they inhibited CaER, and vice versa. Modulating promoter or RBS strength is a common strategy in multi-enzyme cascades to balance protein expression levels. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e) Here, we used four T7 promoter mutants of varying strengths (TA, TT, AA, AT, from strong to weak) in pairwise combinations to fine-tune the expression ratio of FjTAL to CaER. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e) The results showed that the medium-strength AA-AA combination yielded the highest HPPA titer of 26.7 mM, providing ample substrate for the subsequent photodecarboxylation step. After determining the catalytic capacity of each cell catalyst, we used 2 g/L (DCW) of the TAL-ER strain and 10 g/L (DCW) of the FAP strain to convert 5 mM tyrosine from the fermentation broth. Due to the significantly different optimal conditions for the two catalysts, we adopted a sequential reaction strategy. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e) First, 2 g/L (DCW) of the TAL-ER cells completely converted 5 mM tyrosine to p-coumaric acid within 8 hours at 30\u0026deg;C and pH 8.0. Subsequently, 10 g/L (DCW) of the FAP cells were added, and the conditions were adjusted to 16\u0026deg;C and pH 6.5 for a further 2-hour reaction. This process achieved a final 4-EP concentration of 4.02 mM, which is the highest level reported to date.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn summary, this study leverages the innate substrate promiscuity of fatty acid photodecarboxylase (FAP). Through directed evolution, we enhanced its catalytic efficiency for a non-native aromatic substrate by 3.7-fold and identified Y466, G462, P460, and V453 as key engineering hotspots, providing a general strategy for broadening FAP's substrate scope. To achieve de novo synthesis of 4-EP, we constructed a high-yield L-tyrosine chassis (8.6 g/L in shake flasks) following Ping et al., which served as the foundation for the multi-enzyme cascade. Promoter engineering optimized the pathway, elevating the HPPA titer to 26.7 mM. We identified the mismatched pH optima of FjTAL and CaER, along with the oxygen sensitivity of CaER, as the primary bottlenecks for the efficient conversion of L-Tyr to HPPA; this conversion could potentially be further improved by employing acid-tolerant TALs and anaerobic catalysis. Finally, the optimized FAP mutant quantitatively decarboxylated HPPA, yielding a final 4-EP concentration of 4.02 mM\u0026mdash;the highest value reported to date. Our work demonstrates that FAP, with its high efficiency and broad promiscuity, can be integrated as a versatile \"decarboxylation platform module\" into biosynthetic pathways to overcome challenging decarboxylation steps, offering a new paradigm for the green synthesis of aromatic chemicals.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 32100060, 32101229), Fundamental Research Funds for the Central Universities (No. 2662021SKQD001, 2662021JC006), and the LongYun Program 2.0 from College of Life Science and Technology.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGosset, G. 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The enzyme responsible for its biosynthesis, postulated to be a phloretic acid decarboxylase, has not yet been identified in nature. This study unveils that fatty acid photodecarboxylase (FAP) effectively substitutes for this \"missing\" enzyme. Upon photoexcitation, the FAP cofactor directly decarboxylates \u003cem\u003ep\u003c/em\u003e-hydroxyphenyl propionic acid (HPPA) without prerequisite activation, thereby introducing a novel paradigm of light-driven decarboxylation. A semi-rational design yielded a triple FAP mutant (Y466V/G462V/V453M) with 3.7-fold enhanced activity. Leveraging this discovery, we developed an integrated fermentation-photocatalysis process. Engineered E. coli first produced tyrosine from glucose, followed by a two-step whole-cell biotransformation that achieved a record 4-EP titer of 4.02 mM (0.49 g L⁻\u0026sup1;). This cofactor- and toxin-free route establishes a plug-and-play platform for the sustainable synthesis of aromatic compounds.\u003c/p\u003e","manuscriptTitle":"Biosynthesis of 4-ethylphenol via an enzyme cascade with an engineered fatty acid photodecarboxylase","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 09:33:54","doi":"10.21203/rs.3.rs-7714953/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4c28590-7e91-4357-ba00-f69edfc215b9","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":55350867,"name":"Biotechnology and Bioengineering"}],"tags":[],"updatedAt":"2025-09-29T09:33:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 09:33:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7714953","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7714953","identity":"rs-7714953","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}