Vanillin Biosynthesis Research Articles

Pathway-Adapted Biosensor for High-Throughput Screening of O-Methyltransferase and its Application in Vanillin Synthesis.

Vanillin is a widely used flavoring compound in the food, pharmaceutical, and cosmetics area. However, the biosynthesis of vanillin from low-cost shikimic acid is significantly hindered by the low activity of the rate-limiting enzyme, caffeate O-methyltransferase (COMT). To screen COMT variants with improved conversion rates, we designed a biosensing system that is adaptable to the COMT-mediated vanillin synthetic pathway. Through the evolution of aldehyde transcriptional factor YqhC, we obtained a dual-responsive variant, MuYqhC, which positively responds to the product and negatively responds to the substrate, with no response to intermediates. Using the MuYqhC-based vanillin biosensor, we successfully identified a COMT variant, Mu176, that displayed a 7-fold increase in the conversion rate compared to the wild-type COMT. This variant produced 2.38 mM vanillin from 3 mM protocatechuic acid, achieving a conversion rate of 79.33%. The enhanced activity of Mu176 was attributed to an enlarged binding pocket and strengthened substrate interaction. Applying Mu176 to Bacillus subtilis increased the level of vanillin production from shikimic acid by 2.39-fold. Further optimization of the production chassis, increasing the S-adenosylmethionine supply and the precursor concentration, elevated the vanillin titer to 1 mM, marking the highest level of vanillin production from shikimic acid in Bacillus. Our work highlights the significance of the MuYqhC-based biosensing system and the Mu176 variant in vanillin production.

Optimization of vanillin biosynthesis in Escherichia coli K12 MG1655 through metabolic engineering

Vanillin is an important flavouring agent applied in food, spices, pharmaceutical industries and other fields. Microbial biosynthesis of vanillin is considered a sustainable and economically feasible alternative to traditional chemical synthesis. In this study, Escherichia coli K12 MG1655 was used for the de novo synthesis of VAN by screening highly active carboxylic acid reductases and catechol O-methyltransferases, optimising the protocatechuic acid pathway, and regulating competitive metabolic pathways. Additionally, major alcohol by-products were identified and decreased by deleting three endogenous aldo–keto reductases and three alcohol dehydrogenases. Finally, a highest VAN titer was achieved to 481.2 mg/L in a 5 L fermenter from glucose. This work provides a valuable example of pathway engineering and screens several enzyme variants for the first time in E. coli.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.

Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid.

The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants.

Engineering the activity and thermostability of a carboxylic acid reductase in the conversion of vanillic acid to vanillin

Vanillin is a valuable natural product that can be used as a fragrance and additive. Recent research in the biosynthesis of vanillin has brought attention to a key enzyme, carboxylic acid reductase (CAR), which catalyzes the reduction of vanillic acid to vanillin. Nevertheless, the biosynthesis of vanillin is hampered by the low activity and stability of CAR. As such, a rational design campaign was conducted on a well-documented carboxylic acid reductase from Segniliparus rugosus (SrCAR), using vanillic acid as the model substrate. After combined active site saturation and iterative site-specific mutagenesis, the best quadruple mutant N292H/K524S/A627L/E1121W (M3) was successfully obtained. In comparison to the wildtype SrCAR, M3 demonstrated a 4.2-fold increase in catalytic efficiency (kcat/Km), and its half-life (t1/2) was enhanced by 3.8 times up to 385.08 minutes at 40 °C. In silico docking and molecular dynamics simulation provided insights into the improved activity and stability. In the subsequent preparative-scale reaction with 100 mM (16.8 g L−1) vanillic acid, the whole cell catalysis utilizing M3 produced 10.15 g·L−1 of vanillin and 1.11 g·L−1 of vanillyl alcohol, respectively. This work demonstrates a dual improvement in the activity and thermal stability of SrCAR, thereby potentially facilitating the application of carboxylic acid reductase in the biosynthesis of vanillin.

QM/MM Study Into the Mechanism of Oxidative C=C Double Bond Cleavage by Lignostilbene-α,β-Dioxygenase.

The enzymatic biosynthesis of fragrance molecules from lignin fragments is an important reaction in biotechnology for the sustainable production of fine chemicals. In this work we investigated the biosynthesis of vanillin from lignostilbene by a nonheme iron dioxygenase using QM/MM and tested several suggested proposals via either an epoxide or dioxetane intermediate. Binding of dioxygen to the active site of the protein results in the formation of an iron(II)-superoxo species with lignostilbene cation radical. The dioxygenase mechanism starts with electrophilic attack of the terminal oxygen atom of the superoxo group on the central C=C bond of lignostilbene, and the second-coordination sphere effects in the substrate binding pocket guide the reaction towards dioxetane formation. The computed mechanism is rationalized with thermochemical cycles and valence bond schemes that explain the electron transfer processes during the reaction mechanism. Particularly, the polarity of the protein and the local electric field and dipole moments enable a facile electron transfer and an exergonic dioxetane formation pathway.

Engineering yeast to convert lignocellulose into vanillin

Valorizing lignocellulosic biomass (LCB) into platform aromatic chemicals could contribute to a sustainable biomass-based bioeconomy. However, lacking simultaneous bioconversion route of lignin and carbohydrates hinders the LCB utilization. The artificial biological funneling pathway (ABFP) was hence designed to convert full components of LCB into vanillin. The results showed that combinational engineering strategies enabled the successful accumulation of vanillin in Saccharomyces cerevisiae. The funneling pathways of lignin-derived monomers to vanillin were constructed. Enzyme fusion and S-adenosylmethionine metabolism regulation of heterologous rate-limiting enzyme further promoted the vanillin production to 2.49 and 1.94 mmol/L from ferulic acid and p-coumaric acid, respectively. The construction of funneling pathways of hexose and pentose promoted the carbon flux towards vanillin biosynthesis. Together with the regulation of branch and shikimic acid pathways, the microbial cell factory with a novel ABFP produced 1.97 mmol/L vanillin from real LCB hydrolysates with a recorded yield of 10.5 mg vanillin/g carbon source. Overall, the engineered S. cerevisiae simultaneously valorized full components into fine aromatic chemicals, holding the promise to transform biorefinery mode and contribute to the biomass-based economy.

Engineering of halide methyltransferases for synthesis of SAE and its application in biosynthesis of ethyl vanillin

Halide methyltransferase (HMT)-catalyzed synthesis of alkyl donors from S-adenosyl-L-homocysteine (SAH) and haloalkanes is a promising method for biocatalytic alkylation. However, the reported halide methyltransferases show limited catalytic capacity towards unnatural substrates such as ethyl iodide, which limits their application in biocatalytic ethylation. In this work, we performed structure-based directed evolution starting from the wild-type Aspergillus clavatus halide methyltransferase (AcHMT). The best variant, AcHMTM3, was identified with an activity of up to 421.5 mU mg–1 towards ethyl iodide, 38.7-fold higher than that of the wild type AcHMT. Subsequently, molecular dynamics simulations provide structural insights into how mutations improve the catalytic activity towards iodoethane. Finally, AcHMTM3 was utilized in one-pot cascade with the reported O-methyltransferase RnCOMT+Y200L to synthesize ethyl vanillin from 3,4-dihydroxybenzaldehyde (79% conversion). Our work demonstrated the potential of the engineered HMT in further biocatalytic alkylation.

Biosynthesis of Vanillin by Rational Design of Enoyl-CoA Hydratase/Lyase.

Vanillin holds significant importance as a flavoring agent in various industries, including food, pharmaceuticals, and cosmetics. The CoA-dependent pathway for the biosynthesis of vanillin from ferulic acid involved feruloyl-CoA synthase (Fcs) and enoyl-CoA hydratase/lyase (Ech). In this research, the Fcs and Ech were derived from Streptomyces sp. strain V-1. The sequence conservation and structural features of Ech were analyzed by computational techniques including sequence alignment and molecular dynamics simulation. After detailed study for the major binding modes and key amino acid residues between Ech and substrates, a series of mutations (F74W, A130G, A130G/T132S, R147Q, Q255R, ΔT90, ΔTGPEIL, ΔN1-11, ΔC260-287) were obtained by rational design. Finally, the yield of vanillin produced by these mutants was verified by whole-cell catalysis. The results indicated that three mutants, F74W, Q147R, and ΔN1-11, showed higher yields than wild-type Ech. Molecular dynamics simulations and residue energy decomposition identified the basic residues K37, R38, K561, and R564 as the key residues affecting the free energy of binding between Ech and feruloyl-coenzyme A (FCA). The large changes in electrostatic interacting and polar solvating energies caused by the mutations may lead to decreased enzyme activity. This study provides important theoretical guidance as well as experimental data for the biosynthetic pathway of vanillin.

Biosynthesis of vanillin from vanillyl alcohol by recombinant Escherichia coli cells expressing 5-hydroxymethylfurfural oxidase

The efficient valorization of lignin-derived vanillyl alcohol for the production of value-added biovanillin with enzyme or whole-cell based biotransformation is attracting increasing attention. In this study, a recombinant Escherichia coli HMFO was explored for the production of vanillin through the selective aerobic oxidation of vanillyl alcohol. The resting cells of this strain indicated a high vanillin tolerance and good biocatalytic activity over a temperature range of 10–35 °C. The vanillyl alcohol oxidation process produced vanillin as the sole product, indicating excellent reaction selectivity. Vanillyl alcohol substrate of 250 mM was converted to vanillin under optimal conditions with 96 % yield. In addition, 449.2 mM vanillin was obtained in a fed-batch experiment, providing a productivity of 1.2 g/L per hour and 90 % yield. The production of biovanillin could be further improved by using enzyme engineering and process engineering methods.

Current Status, Challenges, and Prospects for the Biological Production of Vanillin

Vanillin has been widely used as a flavoring agent in the food industry and as a precursor in the medicine and polymer industries. However, the use of chemically synthesized vanillin is prohibited in food and some other industries. Additionally, the harsh conditions and toxic substrates in chemically synthesized vanillin lead to some environmental challenges and energy waste. With the rapid development of synthetic biology, the biological production of vanillin from renewable resources through microbial fermentation has gained great attention owing to its high selectivity and environmentally friendly properties. Accordingly, this article will discuss the vanillin biosynthesis technology from the aspects of chassis cell types and substrate types. The key enzymes involved in metabolic pathways are also discussed. Then, we summarize some improvements in the process of vanillin production to increase its production and reduce the toxicity of vanillin in microorganisms, and the possible future directions for vanillin biosynthesis will also be outlined.

De novo biosynthesis of vanillin in engineered Saccharomyces cerevisiae

Vanillin, an important aldehyde compound, is widely used in food and medicine industries. Natural and chemical-synthesized vanillin are facing significant challenge for the sustainabledevelopment.Here, de novo biosynthesis of vanillin inS. cerevisiaewas thus designed. Through integrating sam8, CYP199A2, comt, fcs and echinto the genome of S. cerevisiae, vanillin was successfully biosynthesized with a titer of 26.3 μg/L. Preferred sam5efficiently enriched caffeic acid pool, improving vanillin production to 50.2 μg/L in haploid engineered yeast. Expressing the mutants of ARO4 K229L and ARO7 G141S had reduced the negative feedback of tyrosine, increased the vanillin titer to 700 μg/L. Finally, the titer of vanillin was increased to 8545 μg/L through supplying 400 mg/L ferulic acid. The strategies of metabolic engineering and fermentation optimization achieved de novo biosynthesis of vanillin inS. cerevisiae, showing the feasibility of vanillin production by microbial technology.

Is a spice missing from the recipe? The intra-cellular localization of vanillin biosynthesis needs further investigations.

Vanillin is the most popular flavor compound in the world. Substantial effort were made in the last decades to completely elucidate the metabolic pathway that leads to vanillin in plants, with some controversy reported. In V. planifolia, vanillin biosynthesis occurs in plastids or in redifferentiated-plastids termed ''phenyloplasts''. More recently, it was shown that all enzymes required for the conversion of [14 C]-phenylalanine to [14 C]-vanillin-glucoside are confined within that organelle. However, knowing that some of these enzymes are cytosolic or ER-membrane bound in most plant species, it raises questions on the interpretation of data obtained from the technique used and on the true localization of the biosynthetic enzymes in V.planifolia. In addition, intense debate has emerged about the real participation of last enzyme of the pathway involving vanillin synthase (VpVAN) in the direct conversion of ferulic acid to vanillin. With the discovery of another enzyme capable of this conversion and the lack of activity of VpVAN invitro, further disagreement emerged. One additional challenge to VpVAN being necessary and sufficient is that the transcript for this protein is abundant invarious non-vanillin-producing tissues of the vanilla plant. In this viewpoint, we discuss the findings surrounding the cellular-localization and activity of enzymes of vanillin biosynthesis. This will help to further understand the pathway and urge for additional research study to resolve the debate.

Effective Biotransformation of Variety of Guaiacyl Lignin Monomers Into Vanillin by Bacillus pumilus.

Biotransformation has gained increasing attention due to its being an eco-friendly way for the production of value-added chemicals. The present study aimed to assess the potential of Bacillus pumilus ZB1 on guaiacyl lignin monomers biotransformation for the production of vanillin. Consequently, isoeugenol, eugenol, and vanillyl alcohol could be transformed into vanillin by B. pumilus ZB1. Based on the structural alteration of masson pine and the increase of total phenol content in the supernatant, B. pumilus ZB1 exhibited potential in lignin depolymerization and valorization using masson pine as the substrate. As the precursors of vanillin, 61.1% of isoeugenol and eugenol in pyrolyzed bio-oil derived from masson pine could be transformed into vanillin by B. pumilus ZB1. Four monooxygenases with high specific activity were identified that were involved in the transformation process. Thus, B. pumilus ZB1 could emerge as a candidate in the biosynthesis of vanillin by using wide guaiacyl precursors as the substrates.

Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin.

Vanillin production by metabolic engineering of proprietary microbial strains has gained impetus due to increasing consumer demand for naturally derived products. Here, we demonstrate the use of rice cell cultures metabolically engineered with vanillin synthase gene (VpVAN) as a plant-based alternative to microbial vanillin production systems. VpVAN catalyzes the signature step to convert ferulic acid into vanillin in Vanilla planifolia. As ferulic acid is a phenylpropanoid pathway intermediate in plant cells, rice calli cells are ideal platform for in vivo vanillin synthesis due tothe availability of its precursor. In this study, rice calli derived from embryonic rice cells were metabolically engineered with a codon-optimized VpVAN gene using Agrobacterium-mediated transformation. The putative transformants were selected based on their proliferation on herbicide-supplemented N6D medium. Expression of the transgenes wereconfirmed through a β-glucuronidase (GUS) reporter assay and polymerase chain reaction (PCR) analysis provided evidence of genetic transformation. The semiquantitative RT-PCR and real-time (RT)-qPCR revealed expression of VpVAN in six transgenic calli lines. High-performance liquid chromatography identified the biosynthesis of vanillin in transgenic calli lines, with the highest yielding line producing 544.72 (± 102.50) μg of vanillin-g fresh calli. This work serves as a proof-of-concept to produce vanillin using metabolically engineered rice cell cultures.

Biosynthesis of vanillin by different microorganisms: a review.

Vanillin is a popular flavoring agent widely used around the world. Vanillin is generated by natural extraction, chemical synthesis, or tissue culture technology, but these production methods no longer meet the increasing worldwide demand for vanillin. Accordingly, a biotechnological approach may provide an effective replacement route to obtaining vanillin. Processes for environmentally friendly production of vanillin in microorganisms from different carbon sources, such as eugenol, isoeugenol, lignin, ferulic acid, sugars, and waste residues, with high productivity and yield have been developed. However, challenges remain for optimizing the vanillin biosynthesis process and further improving production titer and yield. In this review, successful and applicable strategies for increasing vanillin titer and yield in different microorganisms are summarized. Additionally, perspectives for further optimizing the production of vanillin are discussed.

Developing efficient vanillin biosynthesis system by regulating feruloyl-CoA synthetase and enoyl-CoA hydratase enzymes.

Vanillin is one of the most commonly used natural-occurring flavors in the world. This study successfully constructed an efficient whole-cell catalytic system for vanillin biosynthesis from ferulic acid by regulating feruloyl-CoA synthetase (FCS) and enoyl-CoA hydratase (ECH). First, we constructed an efficient cell-free catalytic system with FCS-Str (fcs from Streptomyces sp. V-1) and ECH-Str (ech from Streptomyces sp. V-1) combination at 1:1. The efficient cell-free catalytic system provided necessary strategies for optimizing the whole-cell catalytic system. Then, we constructed the recombinant Escherichia coli by heterologously expressing the fcs-Str and ech-Str combination. Moreover, E. coli JM109 was a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. Finally, we first adjusted the ratio of FCS and ECH in E. coli JM109 to 1:1 using two copies of fcs-Str. For higher vanillin production, we further optimized the induction conditions of E. coli JM109 to increase the amount of FCS and ECH. The optimized E. coli JM109-FE-F constructed in this study has the highest vanillin synthesis rate of converting 20mM ferulic acid to 15mM vanillin in 6h among all of the E. coli catalytic systems. Our study made a significant contribution to the construction of the vanillin biosynthesis system and provided a valuable strategy for increasing vanillin production. KEY POINTS: • The efficient cell-free vanillin biosynthesis system was constructed by FCS-Str and ECH-Str combination at 1:1. • Escherichia coli JM109 was determined as a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. • Escherichia coli JM109-FE-F with two copies of fcs-Str and one copy of ech-Str has the highest catalytic efficiency for vanillin production.

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.

Enhanced Thermostability of Pseudomonas nitroreducens Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis

Vanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herein, two rational strategies were combined to improve the thermostability of IEM from Pseudomonas nitroreducens Jin1. Two variants (K83R and K95R) with better thermostability and one mutant (G398A) with higher activity were identified from twenty candidates based on the Surface Residue Replacement method. According to the Consensus Mutagenesis method, one mutant (I352R) with better thermostability and another mutant (L273F) with higher activity were also identified from nine candidates. After combinatorial mutation, a triple mutant K83R/K95R/L273F with the best thermostability and catalytic efficiency was generated. Compared with the wild-type IEM, the thermal inactivation half-lives (t1/2) of K83R/K95R/L273F at 25 °C, 30 °C, and 35 °C increased 2.9-fold, 11.9-fold, and 24.7-fold, respectively. Simultaneously, it also exhibited a 4.8-fold increase in kcat, leading to a 1.2-fold increase in catalytic efficiency (kcat/Km). When the whole cell of K83R/K95R/L273F was applied to the biotransformation of isoeugenol on preparative scale, the vanillin concentration reached 240.1 mM with space-time yield of 109.6 g/L/d, and vanillin was achieved in 77.6% isolated yield and >99% purity.

Valorization of banana peels waste into biovanillin and optimization of process parameters using submerged fermentation

Vanillin is used as an aroma and flavoring agent in the food, beverage, and cosmetic industries. Vanilla planifolia is the major source of natural vanillin with a market price of 1200–4000$ per kg. To fulfill the market demand, low price vanillin is synthesized chemically i.e., 15 $/kg but European and US legislation considers it unnatural and recommends biosynthesis by fermentation. Ferulic acid is the main and costly precursor for vanillin production. It is naturally present in lignin-enriched agricultural wastes. Therefore, the present study was designed to extract ferulic acid from banana peels and then convert it into biovanillin using Enterobacter hormaechei . Different fermentative parameters like substrate concentration, incubation period, pH, temperature, and agitation speed were optimized via submerged fermentation by varying one factor at a time. From 100 g of banana peels, 60 ± 0.35 mg ferulic acid was extracted and used as a substrate for biovanillin production. The maximum significant (p-value < 0.05) production of biovanillin i.e., 3.8 ± 0.14 g/L was observed by bioconversion of 7 g/L of ferulic acid at 24 h of incubation time, pH 7, 30 °C and agitation speed of 150 rpm. Identification of produced vanillin was done by FTIR and quantification by HPLC method. It was found to be 97% pure in comparison to the standard of Sigma-Aldrich. Hence, the fruit by-product i.e., banana peels served as a useful substrate for the biosynthesis of vanillin. These conditions can be further optimized on a pilot scale to commercialize the product. • Ferulic acid is naturally present in lignin enriched agricultural wastes. • Extraction of ferulic acid from banana peels was done and then converted to biovanillin using Enterobacter hormaechei. • Fermentation conditions were optimized via submerged fermentation by varying one factor at a time. • Identification of produced vanillin was done by FTIR and was found to be 97% by HPLC method. • First study on biovanillin production biotechnologically using banana peels as a ferulic acid source.