Vanillyl-alcohol Oxidase Research Articles

Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5± 0.02 Umg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3± 0.5 Umg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for ɣ-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0± 0.1 Umg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

Metasecretome and biochemical analysis of consortium PM-06 during the degradation of nixtamalized maize pericarp

Nixtamalized maize pericarp is an abundant by-product of the industrial processing of maize for tortilla production, and a potential source of prebiotics and biofuels. Consortium PM-06 is a bacterial community with the capacity to efficiently saccharify the nixtamalized pericarp; however, there is scarce information about the enzymatic mechanism involved. In this study, the soluble enzymes secreted by PM-06 in different stages of the degradation process were analyzed using biochemical approaches and label-free quantitative proteomics. Metasecretomic analysis revealed a marked abundance of glycosyl hydrolases in the initial stages of the process, mainly secreted by Bacillus and Paenibacillus. The main function of glycosyl hydrolases was the metabolism of soluble sugars released from maize pericarp. As degradation proceeded, substrate recalcitrance and oxidoreductase mass abundance increased. Actinobacteria, predominantly Microbacterium, secreted a great diversity of oxidoreductases with vanillyl alcohol oxidase as the most abundant enzyme. Lignin consumption in the last 48 h of culture and the lack of laccase and peroxidase sequences, suggest a role for Fenton chemistry. Different oxidoreductases secreted by PM-06 produce hydrogen peroxide, a co-substrate for various enzymes and a Fenton reactant. A strategy used by PM-06 to cope with substrate recalcitrance could be the use of hydroxyl radicals produced from H2O2 to attack polysaccharides and lignin. This study is the first report about a consortium secreting a great diversity of soluble oxidoreductases and provides information about possible synergistic interactions and functions of bacterial enzymes involved in lignocellulose degradation.

Analyses of the efficiency of a recombinant vanillyl alcohol oxidase in producing vanillin in the hydrothermal liquefaction aqueous phase

In hydrothermal liquefaction (HTL) of biomass, only the oil and the char phases are considered imperative while the aqueous phase is discarded. However, the aqueous phase is rich in phenolic compounds like guaiacol and vanillyl alcohol that can be upgraded to vanillin. Commercially vanillin is synthesized from phenolic compounds derived from fossil fuels using chemical synthesis methods. In this investigation, the method was developed to use raw materials and enzymatic conversion of substrates which is sustainable and renewable. The purpose of this investigation is to evaluate the applicability of enzymatic vanillin synthesis within the HTL aqueous phase major components. Vanillyl alcohol oxidase was expressed in E. coli BL21 (DE3) bacteria cells and purified to characterize the production of vanillin from vanillyl alcohol. The effect on the enzyme reaction of each of the major components of the aqueous phase as detected by HPLC (High Performance Liquid Chromatography) analysis was evaluated. The components evaluated in this study included different phenolic compounds namely guaiacol, vanillic acid, vanillyl alcohol, phenol, catechol and acetic acid. These components were evaluated individually as well as in different combinations. Vanillic acid increased the reaction rate while guaiacol inhibited the reaction rate. Acetic acid exhibited the largest inhibition of the enzyme reaction after 15 min. It is known that acids are often used to stop enzyme reactions as it results in precipitation of the enzyme. Therefore, to successfully convert vanillin from vanillyl alcohol using a recombinantly expressed vanillyl alcohol oxidase, the acetic acid would first need to be removed from the HTL aqueous phase.

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.

Efficient vanillin biosynthesis by recombinant lignin-degrading bacterium Arthrobacter sp. C2 and its environmental profile via life cycle assessment

Vanillin is a natural flavoring agent that is widely used in the bioengineering industry. To enable sustainable development, joint consideration of bacterial performance and negative environmental impacts are critical to vanillin biosynthesis. In this study, a cold shock protein (csp) gene was upregulated for maintaining stable growth in Arthrobacter sp. C2 responding to vanillin and cold stress. Furthermore, the recombinant strain C2 was constructed by simultaneously deleting the xylC gene encoding benzaldehyde dehydrase and overexpressing the pchF gene encoding vanillyl alcohol oxidase and achieved a maximum vanillin productivity of 0.85 mg/g DCW/h with alkaline lignin as the substrate. Finally, this process generated an environmental impact value of 25.05, which was the lowest environmental impact achieved according to life cycle assessment (LCA). Improvement strategies included reducing electricity consumption and replacing chemicals. This study achieved the development of an effective strategy, and future studies should focus on precise vanillin biosynthesis methods for large-scale application.

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

The vanillyl‐alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO‐homolog featuring a glutamate residue in place of the common aspartate. This 4‐ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α‐N3‐histidyl‐bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para‐substituted phenols with little generation of hydroxylated products. The three‐dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.

Characteristics and functional bacteria in a microbial consortium for rice straw lignin-degrading

The degradation of lignin is the main rate-limiting step in the bio-pulping of rice straw. A lignin-degrading bacterial consortium LDC, which can efficiently degrade lignin of reed, was screened in the early stage of our laboratory work. In present study, 7-day incubation of LDC can degrade rice straw lignin by 31.18% in mineral salt medium. The communities’ structure of different incubation phases varied greatly, in which high abundance (44.78%) of Anaerocolumna was first found. The expression levels of lignin degradation enzyme class II peroxidase (AA2), vanillyl alcohol oxidase (AA4) and 1,4-benzoquinone reductase (AA6) during peak phase (48 h) were significantly up-regulated than initial phase (24 h), increasing by 112%, 165% and 67%, respectively, and 42.86% AA2 was from Thaurea; 100% AA4 was from Clostridium; 62.5% AA6 was from Pseudomonas. These provide microbial resources and data support for the industrialization of rice straw bio-pulping.

Improving bioconversion of eugenol to coniferyl alcohol by constitutive promoters in Escherichia coli

Coniferyl alcohol is a common monolignol, which is a precursor of lignin. Coniferyl alcohol is rarely found as free molecules in plants based on its conversion to lignins by peroxidases or laccases. It can be used for biosynthesis of silybin and a lot of pinoresinol derivatives. Although increased output of coniferyl alcohol has been attempted, few methods are effective. In this study, we evaluated 20 promoters for their ability to express genes in Penicillium simplicissimum encoding vanillyl alcohol oxidase (PsVAO) and catalase (ScCTA1) for producing coniferyl alcohol, while eliminating harmful H2O2. We constructed 100 combinations of constitutive promoters in E. coli to enhance gene expression. Then orthogonal experiments, fed-batch kinetic model and CRISPR/Cas9 system were followed to improve the yield of coniferyl alcohol, while eliminate the pinoresinol. Finally, the coniferyl alcohol titers, bioconversion rates, and productivity levels reaching 53.90 g/L, 86.72 %, and 2.45 g/(L × h), respectively.

Cloning, expression and enzymatic characterization of recombinant eugenol oxidase (EUGO) in Escherichia coli

Eugenol oxidase (EUGO), a member of the vanillyl alcohol oxidase family, catalyzes the oxidative reaction of vanillyl alcohol to vanillin. This compound is responsible for the vanilla aroma and is widely used as a flavoring agent in food, cosmetics, and pharmaceuticals. Previously, EUGO was cloned and expressed in E. coli TOP10, and purified by anion-exchange chromatography with Q-Sepharose resin but the purification factor was low. To improve the efficiency of the EUGO purification, in this study, we cloned eugo gene into pET-28a vector and expressed it in E. coli Tunetta. The SDS-PAGE analysis of protein extracts obtained from E. coli expressing EUGO under different induction conditions showed that EUGO was expressed mostly in the soluble fraction at 6 hours after induction with 0.1 mM IPTG at 25oC. EUGO was purified by immobilized−metal affinity chromatography with Ni2+-NTA agarose and the in vitro enzymatic activity was characterized. The specific activity of purified EUGO was nearly 4-fold higher than that of the crude enzyme sample. In particular, the enzyme preparation produced by the purification method based on Ni-NTA affinity in this study was 2,5-fold more pure than that produced by Q-sepharose purification method described previously.

Cholesterol oxidase from Rhodococcus erythropolis with high specificity toward β-cholestanol and pytosterols.

Two genes (choRI and choRII) encoding cholesterol oxidases belonging to the vanillyl-alcohol oxidase (VAO) family were cloned on the basis of putative cholesterol oxidase gene sequences in the genome sequence data of Rhodococcus erythropolis PR4. The genes corresponding to the mature enzymes were cloned in a pET vector and expressed in Escherichia coli. The two cholesterol oxidases produced from the recombinant E. coli were purified to examine their properties. The amino acid sequence of ChoRI showed significant similarity (57%) to that of ChoRII. ChoRII was more stable than ChoRI in terms of pH and thermal stability. The substrate specificities of these enzymes differed distinctively from one another. Interestingly, the activities of ChoRII toward β-cholestanol, β-sitosterol, and stigmasterol were 2.4-, 2.1-, and 1.7-fold higher, respectively, than those of cholesterol. No cholesterol oxidases with high activity toward these sterols have been reported so far. The cholesterol oxidation products from these two enzymes also differed. ChoRI and ChoRII oxidized cholesterol to form cholest-4-en-3-one and 6β-hydroperoxycholest-4-en-3-one, respectively.

Vanillyl alcohol oxidase.

This review presents a historical outline of the research on vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum, one of the canonical members of the VAO/PCMH flavoprotein family. After describing its discovery and initial biochemical characterization, we discuss the physiological role, substrate scope, and catalytic mechanism of VAO, and review its three-dimensional structure and mechanism of covalent flavinylation. We also explain how protein engineering provided a deeper insight into the role of certain amino acid residues in determining the substrate specificity and enantioselectivity of the enzyme. Finally, we summarize recent computational studies about the migration of substrates and products through the enzyme's structure and the phylogenetic distribution of VAO and related enzymes.

Mechanism of the Flavoprotein d-6-Hydroxynicotine Oxidase: Substrate Specificity, pH and Solvent Isotope Effects, and Roles of Key Active-Site Residues.

The flavoprotein d-6-hydroxynicotine oxidase catalyzes an early step in the oxidation of ( R)-nicotine, the oxidation of a carbon-nitrogen bond in the pyrrolidine ring of ( R)-6-hydroxynicotine. The enzyme is a member of the vanillyl alcohol oxidase/ p-cresol methylhydroxylase family of flavoproteins. The effects of substrate modifications on the steady-state and rapid-reaction kinetic parameters are not consistent with the quinone-methide mechanism of p-cresol methylhydroxylase. There is no solvent isotope effect on the kcat/ Kamine value with either ( R)-6-hydroxynicotine or the slower substrate ( R)-6-hydroxynornicotine. The effect of pH on the rapid-reaction kinetic parameters establishes that only the neutral form of the substrate and the correctly protonated form of the enzyme bind. The active-site residues Lys348, Glu350, and Glu352 are all properly positioned for substrate binding. The K348M substitution has only a small effect on the kinetic parameters; the E350A and E350Q substitutions decrease the kcat/ Kamine value by ∼20- and ∼220-fold, respectively, and the E352Q substitution decreases this parameter ∼3800-fold. The kcat/ Kamine-pH profile is bell-shaped. The p Ka values in that profile are altered by replacement of ( R)-6-hydroxynicotine with ( R)-6-hydroxynornicotine as the substrate and by the substitutions for Glu350 and Glu352, although the profiles remain bell-shaped. The results are consistent with a network of hydrogen-bonded residues in the active site being involved in binding the neutral form of the amine substrate, followed by the transfer of a hydride from the amine to the flavin.

An Enzyme Cascade Synthesis of Vanillin

A novel approach for the synthesis of vanillin employing a three-step two-enzymatic cascade sequence is reported. Cytochrome P450 monooxygenases are known to catalyse the selective hydroxylation of aromatic compounds, which is one of the most challenging chemical reactions. A set of rationally designed variants of CYP102A1 (P450 BM3) from Bacillus megaterium at the amino acid positions 47, 51, 87, 328 and 437 was screened for conversion of the substrate 3-methylanisole to vanillyl alcohol via the intermediate product 4-methylguaiacol. Furthermore, a vanillyl alcohol oxidase (VAO) variant (F454Y) was selected as an alternative enzyme for the transformation of one of the intermediate compounds via vanillyl alcohol to vanillin. As a proof of concept, the bi-enzymatic three-step cascade conversion of 3-methylanisole to vanillin was successfully evaluated both in vitro and in vivo.

Improving bioconversion of eugenol to coniferyl alcohol by in situ eliminating harmful H2O2

Coniferyl alcohol is a valuable chemical. However, the current approaches to obtain coniferyl alcohol are either unefficient or expensive. Penicillium simplicissimum vanillyl alcohol oxidase (PsVAO) can be used to produce coniferyl alcohol. However, PsVAO intrinsically produces harmful byproduct H2O2. Utilizing catalase to decompose H2O2 is a potential straightforward approach; however, catalase can also exhibit peroxidase activity to facilitate coniferyl alcohol over-oxidation. In this study, catalases exhibiting both high catalase activity and low peroxidase activity were found out, and introduced into the bioconversion systems. Our results showed that eliminating H2O2in situ released H2O2 inhibition of PsVAO, improved coniferyl alcohol production and eliminated coniferyl alcohol over-oxidation. Finally, coniferyl alcohol titer, molar yield, and productivity reached 22.9 g/L, 78.7%, and 0.5 g/(L × h) respectively. An efficient coniferyl alcohol production method was developed by overcoming the intrinsic disadvantages of PsVAO.

Multigram Scale Enzymatic Synthesis of (R)‐1‐(4′‐Hydroxyphenyl)ethanol Using Vanillyl Alcohol Oxidase

AbstractThe enantioselective oxyfunctionalisation of C−H bonds is a highly interesting reaction, as it provides access to chiral alcohols that are important pharmaceutical building blocks. However, it is hard to achieve using traditional methods. One way in which it can be achieved is through the action of oxidative enzymes. Although many reports of the oxyfunctionalisation capabilities of enzymes at an analytical scale have been published, reports on the use of enzymes to achieve oxyfunctionalisation on a synthetically relevant scale are fewer. Here, we describe the scale‐up of the conversion of 4‐ethylphenol to (R)‐1‐(4′‐hydroxyphenyl)ethanol using the flavin‐dependent enzyme vanillyl alcohol oxidase. The process was optimised by testing different reaction media and substrate and enzyme concentrations and by performing it under an oxygen atmosphere. Under optimised reaction conditions, 4.10 g (R)‐1‐(4′‐hydroxyphenyl)ethanol at 97% ee was obtained from 10 g 4‐ethylphenol (isolated yield 36%). These results highlight some of the challenges that can be encountered during scale‐up of an enzymatic oxyfunctionalisation process to a synthetically relevant scale and will be of use for the development of enzymatic processes for the synthesis of industrially relevant compounds.magnified image

On the origin of vanillyl alcohol oxidases

Vanillyl alcohol oxidase (VAO) is a fungal flavoenzyme that converts a wide range of para-substituted phenols. The products of these conversions, e.g. vanillin, coniferyl alcohol and chiral aryl alcohols, are of interest for several industries. VAO is the only known fungal member of the 4-phenol oxidising (4PO) subgroup of the VAO/PCMH flavoprotein family. While the enzyme has been biochemically characterised in great detail, little is known about its physiological role and distribution in fungi.We have identified and analysed novel, fungal candidate VAOs and found them to be mostly present in Pezizomycotina and Agaricomycotina. The VAOs group into three clades, of which two clades do not have any characterised member. Interestingly, bacterial relatives of VAO do not form a single outgroup, but rather split up into two separate clades.We have analysed the distribution of candidate VAOs in fungi, as well as their genomic environment. VAOs are present in low frequency in species of varying degrees of relatedness and in regions of low synteny. These findings suggest that fungal VAOs may have originated from bacterial ancestors, obtained by fungi through horizontal gene transfer.Because the overall conservation of fungal VAOs varies between 60 and 30% sequence identity, we argue for a more reliable functional prediction using critical amino acid residues. We have defined a sequence motif P-x-x-x-x-S-x-G-[RK]-N-x-G-Y-G-[GS] that specifically recognizes 4PO enzymes of the VAO/PCMH family, as well as additional motifs that can help to further narrow down putative functions. We also provide an overview of fingerprint residues that are specific to VAOs.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para-phenol oxidases, facilitating the enzyme engineering of known para-phenol oxidases and the evaluation of the substrate specificity of novel para-phenol oxidases.

Statistically optimized production and characterization of vanillin from creosol using newly isolated Klebsiella pneumoniae P27

The current research study deals with the screening of a potent vanillin-producing microorganism among 96 isolated strains. Biochemical characterization and molecular identification confirmed that the isolated strain belongs to the Klebsiella pneumoniae bacteria, so it was denoted as Klebsiella pneumoniae P27. The optimization of medium components for the enhanced production of vanillin was carried out using two-stage statistical experimental designs, in which the significant medium components for vanillin production were screened using a Plackett-Burman experimental design. And the optimal levels of those noteworthy factors were determined by using central composite design. The statistical optimization of medium components resulted in increases in vanillin production and vanillyl alcohol oxidase activity of 2.05-fold and 3.055-fold, respectively. The highest vanillin production (30.88 mg/L) and vanillyl alcohol oxidase activity (0.044 U/mL) was observed after 16 h of incubation in the presence of 0.26 mL/L creosol, 8.06 g/L yeast extract and 2.77 g/L NH4NO3 in the production medium. The optimally produced vanillin was extracted and confirmed using FTIR and LCMS spectral analysis. The results of the current study support a statistical process optimization approach as a potential technique for the enhanced production of vanillin from creosol by using newly isolated Klebsiella pneumoniae P27 bacterial strain.

Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase

A number of oxidoreductases from the VAO/para-cresol methylhydroxylase flavoprotein family catalyze the oxidation of para-substituted phenols. One of the best-studied is vanillyl-alcohol oxidase (VAO) from the fungus Penicillium simplicissimum. For oxidation of phenols by VAO to occur, they must first be bound in the active site of the enzyme in their phenolate anion form. The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation. To investigate their role in catalysis, we created three VAO variants, Y108F, Y503F, and Y108F/Y503F, and studied their biochemical properties. Steady-state kinetics indicated that the presence of at least one of the tyrosine residues is essential for efficient catalysis by VAO. Stopped-flow kinetics revealed that the reduction of VAO by chavicol or vanillyl alcohol occurs at two different rates: kobs1, which corresponds to its reaction with the deprotonated form of the substrate, and kobs2, which corresponds to its reaction with the protonated form of the substrate. In Y108F, Y503F, and Y108F/Y503F, the relative contribution of kobs2 to the reduction is larger than in wild-type VAO, suggesting deprotonation is impaired in these variants. Binding studies disclosed that the competitive inhibitor isoeugenol is predominantly in its deprotonated form when bound to wild-type VAO, but predominantly in its protonated form when bound to the variants. These results indicate that Tyr-108 and Tyr-503 are responsible for the activation of substrates in VAO, providing new insights into the catalytic mechanism of VAO and related enzymes that oxidize para-substituted phenols.

Engineering of an H2 O2 auto-scavenging in vivo cascade for pinoresinol production.

Pinoresinol is a natural lignan with a high market value that has potential pharmacological and food supplement applications. Pinoresinol is currently isolated from plants, which suffers from low efficiency and yield. To produce pinoresinol from inexpensive and industrially available eugenol, an in vivo enzymatic cascade composed of vanillyl alcohol oxidase and peroxidase was designed, which scavenges H2 O2 automatically and eliminates protein purification and cofactor addition. Two peroxidases were screened and identified from Escherichia coli BL21 (DE3), and tested in the enzymatic cascade. To balance the flux, different genetic architectures were constructed by using ePathBrick and fusion gene approaches. Scavenging H2 O2 alleviated by-product toxicity and enzyme inhibition, and led to efficient pinoresinol production. Optimization of the reaction conditions achieved a titer of 11.29 g/L pinoresinol. The molar yield and productivity were 52.77% and 1.03 g/(L × h), respectively. The elegant strategy developed herein utilizes the harmful by-product to drive the biosynthetic reaction forward and simultaneously detoxify cells, thereby preventing enzyme inhibition. Biotechnol. Bioeng. 2017;114: 2066-2074. © 2017 Wiley Periodicals, Inc.