Phenolic Acid Decarboxylase Research Articles

One-pot bioconversion of lignin to 4-vinylphenol derivatives

Lignin, the largest renewable aromatic resource in nature, is a promising feedstock to produce aromatic fine chemicals. The bioconversion of lignin into 4-vinylphenol derivatives (4VPs) is a sustainable route to promote both lignin valorization and bioeconomy. However, challenges such as heterologous lignin and an inefficient synthetic pathway for 4VPs restrict the bioconversion performance. In this work, microbial cell factories were designed to valorize lignin into 4VPs, emphasizing an atom-economic route. Rewriting endogenous pathways and heterogeneously expressing phenolic acid decarboxylases successfully produced 4VPs from lignin-derived aromatic precursors. Enzymatic kinetics revealed that the screened phenolic acid decarboxylases possessed outstanding catalytic activity against lignin derivatives. A biphasic glyceryl tributyrate/water system was developed for efficient in situ extraction of 4VPs, successfully removing the product-feedback inhibition effect. Accordingly, the microbial cell factory produced the record titers of 4-vinylphenol, 4-vinylguaiacol and 4-vinylsyringol at 235.3, 203.0 and 35.8 g/L, respectively, with the productivity of 3.0, 2.7, and 0.8 g/L/h. The microbial cell factory was further engineered to simultaneously convert three-type aromatic of lignin into 4VPs through a one-pot bioconversion. The microbial cell factory also exhibited the remarkable bioconversion capacity of actual lignin hydrolysate by producing 12.4 g/L and 2.3 g/L of 4-vinylphenol and 4-vinylguaiacol, respectively. The molecular mechanism suggested that corresponding polymers were successfully produced and displayed good thermal stability. Overall, the microbial cell factory demonstrated an impressive capacity to convert lignin derivatives and achieve record titers of 4VPs, showcasing its potential to revolutionize lignin valorization and the production of functional polystyrene materials.

Characterization of Brettanomyces bruxellensis Phenolic Acid Decarboxylase Enzyme Expressed in Escherichia coli

Brettanomyces yeasts are considered spoilage organisms in commercial fermentation settings; however, some historical beer styles and several modern specialty beer styles rely on Brettanomyces for unique aroma and flavor contributions. The organoleptic properties of beer utilizing intentional Brettanomyces fermentations can be unpredictable and inconsistent. This study investigates the contributions of Brettanomyces fermentations to volatile phenolic character by expression of phenolic acid decarboxylase enzyme cloned from B. bruxellensis. Protein was expressed and isolated from Escherichia cells. The enzyme has broad temperature and pH ranges, and showed variable activity against the most common potential hydroxycinnamic acid substrates.

Integrated preservation of water activity as key to intensified chemoenzymatic synthesis of bio-based styrene derivatives

The valorization of lignin-derived feedstocks by catalytic means enables their defunctionalization and upgrading to valuable products. However, the development of productive, safe, and low-waste processes remains challenging. This paper explores the industrial potential of a chemoenzymatic reaction performing the decarboxylation of bio-based phenolic acids in wet cyclopentyl methyl ether (CPME) by immobilized phenolic acid decarboxylase from Bacillus subtilis, followed by a base-catalyzed acylation. Key-to-success is the continuous control of water activity, which fluctuates along the reaction progress, particularly at high substrate loadings (triggered by different hydrophilicities of substrate and product). A combination of experimentation, thermodynamic equilibrium calculations, and MD simulations revealed the change in water activity which guided the integration of water reservoirs and allowed process intensification of the previously limiting enzymatic step. With this, the highly concentrated sequential two-step cascade (400 g·L–1) achieves full conversions and affords products in less than 3 h. The chemical step is versatile, accepting different acyl donors, leading to a range of industrially sound products. Importantly, the finding that water activity changes in intensified processes is an academic insight that might explain other deactivations of enzymes when used in non-conventional media.

Photokatalytische CO2 Reduktion mit CO2‐bindenden Enzymen

AbstractNeuartige Konzepte zur Nutzung von Kohlendioxid sind erforderlich, um eine kreislauforientierte Kohlenstoffwirtschaft zu erreichen und Umweltprobleme zu minimieren. Um diese Ziele zu erreichen, sind Photo‐, Elektro‐, thermische und Biokatalyse die Schlüsselwerkzeuge, die vorzugsweise in wässrigen Lösungen eingesetzt werden. Dennoch gibt es nur wenige katalytische Systeme, die in Wasser effizient arbeiten. Hier stellen wir eine allgemeine Strategie zur Identifizierung von Enzymen vor, die für die CO2‐Reduktion geeignet sind und zwar auf der Grundlage von Strukturanalysen für potenzielle Kohlendioxid‐Bindungsstellen und anschließenden Mutationen. Wir entdeckten, dass die Phenolsäure‐Decarboxylase aus Bacillus subtilis (BsPAD) die wässrige photokatalytische CO2‐Reduktion in Gegenwart eines Ruthenium‐Photosensibilisators und Natriumascorbat selektiv zu Kohlenmonoxid‐Bildung ermöglicht. Mit modifizierten Varianten von BsPAD wurden TONs von bis zu 978 und Selektivitäten von bis zu 93 % (wobei die gewünschte CO‐ gegenüber der H2‐Erzeugung bevorzugt wurde) erreicht. Durch Mutationen im Bereich des aktiven Zentrums von BsPAD wurden die Umsatzzahlen für die CO‐Erzeugung weiter verbessert. Dabei zeigte sich, dass der Elektronentransfer geschwindigkeitsbegrenzend ist und durch mehrstufiges Tunneln erfolgt. Die Allgemeingültigkeit dieses Ansatzes wurde durch die Verwendung von acht anderen Enzymen bewiesen, die alle die gewünschte Aktivität zeigten, was unterstreicht, dass eine Reihe von Proteinen zur photokatalytischen CO2‐Reduktion in der Lage ist.

Photocatalytic CO2 Reduction Using CO2-Binding Enzymes.

Novel concepts to utilize carbon dioxide are required to reach a circular carbon economy and minimize environmental issues. To achieve these goals, photo-, electro-, thermal-, and biocatalysis are key tools to realize this, preferentially in aqueous solutions. Nevertheless, catalytic systems that operate efficiently in water are scarce. Here, we present a general strategy for the identification of enzymes suitable for CO2 reduction based on structural analysis for potential carbon dioxide binding sites and subsequent mutations. We discovered that the phenolic acid decarboxylase from Bacillus subtilis (BsPAD) promotes the aqueous photocatalytic CO2 reduction selectively to carbon monoxide in the presence of a ruthenium photosensitizer and sodium ascorbate. With engineered variants of BsPAD, TONs of up to 978 and selectivities of up to 93 % (favoring the desired CO over H2 generation) were achieved. Mutating the active site region of BsPAD further improved turnover numbers for CO generation. This also revealed that electron transfer is rate-limiting and occurs via multistep tunneling. The generality of this approach was proven by using eight other enzymes, all showing the desired activity underlining that a range of proteins is capable of photocatalytic CO2 reduction.

Stability Increase of Phenolic Acid Decarboxylase by a Combination of Protein and Solvent Engineering Unlocks Applications at Elevated Temperatures.

Enzymatic decarboxylation of biobased hydroxycinnamic acids gives access to phenolic styrenes for adhesive production. Phenolic acid decarboxylases are proficient enzymes that have been applied in aqueous systems, organic solvents, biphasic systems, and deep eutectic solvents, which makes stability a key feature. Stabilization of the enzyme would increase the total turnover number and thus reduce the energy consumption and waste accumulation associated with biocatalyst production. In this study, we used ancestral sequence reconstruction to generate thermostable decarboxylases. Investigation of a set of 16 ancestors resulted in the identification of a variant with an unfolding temperature of 78.1 °C and a half-life time of 45 h at 60 °C. Crystal structures were determined for three selected ancestors. Structural attributes were calculated to fit different regression models for predicting the thermal stability of variants that have not yet been experimentally explored. The models rely on hydrophobic clusters, salt bridges, hydrogen bonds, and surface properties and can identify more stable proteins out of a pool of candidates. Further stabilization was achieved by the application of mixtures of natural deep eutectic solvents and buffers. Our approach is a straightforward option for enhancing the industrial application of the decarboxylation process.

A New Phenolic Acid Decarboxylase from the Brown-Rot Fungus Neolentinus lepideus Natively Decarboxylates Biosourced Sinapic Acid into Canolol, a Bioactive Phenolic Compound.

Rapeseed meal (RSM) is a cheap, abundant and renewable feedstock, whose biorefinery is a current challenge for the sustainability of the oilseed sector. RSM is rich in sinapic acid (SA), a p-hydroxycinnamic acid that can be decarboxylated into canolol (2,6-dimethoxy-4-vinylphenol), a valuable bioactive compound. Microbial phenolic acid decarboxylases (PADs), mainly described for the non-oxidative decarboxylation of ferulic and p-coumaric acids, remain very poorly documented to date, for SA decarboxylation. The species Neolentinus lepideus has previously been shown to biotransform SA into canolol in vivo, but the enzyme responsible for bioconversion of the acid has never been characterized. In this study, we purified and characterized a new PAD from the canolol-overproducing strain N. lepideus BRFM15. Proteomic analysis highlighted a sole PAD-type protein sequence in the intracellular proteome of the strain. The native enzyme (NlePAD) displayed an unusual outstanding activity for decarboxylating SA (Vmax of 600 U.mg-1, kcat of 6.3 s-1 and kcat/KM of 1.6 s-1.mM-1). We showed that NlePAD (a homodimer of 2 × 22 kDa) is fully active in a pH range of 5.5-7.5 and a temperature range of 30-55 °C, with optima of pH 6-6.5 and 37-45 °C, and is highly stable at 4 °C and pH 6-8. Relative ratios of specific activities on ferulic, sinapic, p-coumaric and caffeic acids, respectively, were 100:24.9:13.4:3.9. The enzyme demonstrated in vitro effectiveness as a biocatalyst for the synthesis of canolol in aqueous medium from commercial SA, with a molar yield of 92%. Then, we developed processes to biotransform naturally-occurring SA from RSM into canolol by combining the complementary potentialities of an Aspergillus niger feruloyl esterase type-A, which is able to release free SA from the raw meal by hydrolyzing its conjugated forms, and NlePAD, in aqueous medium and mild conditions. NlePAD decarboxylation of biobased SA led to an overall yield of 1.6-3.8 mg canolol per gram of initial meal. Besides being the first characterization of a fungal PAD able to decarboxylate SA, this report shows that NlePAD is very promising as new biotechnological tool to generate biobased vinylphenols of industrial interest (especially canolol) as valuable platform chemicals for health, nutrition, cosmetics and green chemistry.

Flow‐Induced Microfluidic Assembly for Advanced Biocatalysis Materials

AbstractExploring the potential of microfluidic systems, this study presents a groundbreaking approach harnessing energy in microfluidic flows within a purpose‐built microreactor, enabling precise deposition of functional biomaterials. Upon optimizing reactor dimensions and integrating it into a microfluidic system, sequentially flow‐induced deposition of DNA hydrogels and transformation into DNA‐protein hybrid materials with SpyTag/SpyCatcher technology is investigated. However, limited functionalization rates restrict its viability for targeted biocatalytic processes. Therefore, the direct deposition of a phenolic acid decarboxylase is investigated, which is efficiently deposited but shows limited biocatalytic performance due to shear‐induced denaturation. This challenge is overcome by a two‐step immobilization process, resulting in microfluidic bioreactors demonstrating initial high space‐time yields of up to 7000 g L−1 d−1, but whose process stability proves unsatisfactory. However, by exploiting the principle of flow‐induced deposition to immobilize recombinant E. coli cells as functional living materials overexpressing biocatalytically relevant enzymes, bioreactors are produced that show equally high space‐time yields in continuous whole‐cell catalysis which remain constant over periods of up to 10 days. The insights gained offer optimization strategies for advanced functional materials and innovative reactor systems holding promise for applications in fundamental materials science, biosensing, and scalable production of microreactors for biocatalysis and bioremediation.

Effects of Fermentation on Bioactivity and the Composition of Polyphenols Contained in Polyphenol-Rich Foods: A Review.

Polyphenols, as common components with various functional activities in plants, have become a research hotspot. However, researchers have found that the bioavailability and bioactivity of plant polyphenols is generally low because they are usually in the form of tannins, anthocyanins and glycosides. Polyphenol-rich fermented foods (PFFs) are reported to have better bioavailability and bioactivity than polyphenol-rich foods, because polyphenols are used as substrates during food fermentation and are hydrolyzed into smaller phenolic compounds (such as quercetin, kaempferol, gallic acid, ellagic acid, etc.) with higher bioactivity and bioavailability by polyphenol-associated enzymes (PAEs, e.g., tannases, esterases, phenolic acid decarboxylases and glycosidases). Biotransformation pathways of different polyphenols by PAEs secreted by different microorganisms are different. Meanwhile, polyphenols could also promote the growth of beneficial bacteria during the fermentation process while inhibiting the growth of pathogenic bacteria. Therefore, during the fermentation of PFFs, there must be an interactive relationship between polyphenols and microorganisms. The present study is an integration and analysis of the interaction mechanism between PFFs and microorganisms and is systematically elaborated. The present study will provide some new insights to explore the bioavailability and bioactivity of polyphenol-rich foods and greater exploitation of the availability of functional components (such as polyphenols) in plant-derived foods.

Spectrophotometric and fluorimetric high-throughput assays for phenolic acid decarboxylase.

Biocatalytic decarboxylation of hydroxycinnamic acids yields phenolic styrenes, which are important precursors for antioxidants, epoxy coatings, adhesives and other polymeric materials. Bacillus subtilis decarboxylase (BsPAD) is a cofactor-independent enzyme that catalyzes the cleavage of carbon dioxide from p-coumaric-, caffeic-, and ferulic acid with high catalytic efficiency. Real-time spectroscopic assays for decarboxylase reactions remove the necessity of extensive sample workup, which is required for HPLC, mass spectrometry, gas chromatography, or NMR methods. This work presents two robust and sensitive assays based on photometry and fluorimetry that allow following decarboxylation reactions with high sensitivity while avoiding product extraction and long analysis times. Optimized assay procedures were used to measure BsPAD activity in cell lysates and to determine the kinetic constants (KM and Vmax) of the purified enzyme for p-coumaric-, caffeic- and ferulic acid. Substrate inhibition was shown for caffeic acid.

Cloning and characterization of phenolic acid decarboxylase responsible for aromatic volatile phenols production in Paocai based on metatranscriptomics

Sichuan Paocai is a kind of fermented vegetable with rich microorganism and enzyme resources. Based on our previous metatranscriptomics and flavor-oriented metabolomics studies, three potential phenolic acid decarboxylases LvPAD, DhFDC1 and LvUbiD were mined from environmental RNA of industrial Sichuan Paocai, and their catalytic function were verified by heterologous expression. LvPAD, which was derived from Lactobacillus vermolensis and had the highest catalytic activity among the three enzymes, was selected for further purification and characterization. It was optimally active at pH 6.0 and 35 °C, had good thermal stability and pH tolerance, and did not require specific cofactors. Kinetic parameters showed that LvPAD could catalyze the decarboxylation of p-coumaric acid, ferulic acid and caffeic acid with the kcat/Km values of 460.6 ± 0.1, 23.7 ± 0.09 and 118.2 ± 0.1 mM−1 s−1, respectively, and the products of the first two substrates were 4-vinylphenol and 4-vinylguaiacol with aromatic characteristics. Moreover, LvPAD was successfully used to preliminarily strengthen the vinylphenol aroma of industrial Paocai, which could shorten the fermentation time to a certain extent in practical production. Therefore, this work not only validated the important role of LvPAD in the production of aromatic phenols in Paocai, but also facilitated the mining of more enzymes from Paocai to improve the flavor and production process in the future.

The Quantum Chemical Cluster Approach in Biocatalysis

The quantum chemical cluster approach has been used for modeling enzyme active sites and reaction mechanisms for more than two decades. In this methodology, a relatively small part of the enzyme around the active site is selected as a model, and quantum chemical methods, typically density functional theory, are used to calculate energies and other properties. The surrounding enzyme is modeled using implicit solvation and atom fixing techniques. Over the years, a large number of enzyme mechanisms have been solved using this method. The models have gradually become larger as a result of the faster computers, and new kinds of questions have been addressed. In this Account, we review how the cluster approach can be utilized in the field of biocatalysis. Examples from our recent work are chosen to illustrate various aspects of the methodology. The use of the cluster model to explore substrate binding is discussed first. It is emphasized that a comprehensive search is necessary in order to identify the lowest-energy binding mode(s). It is also argued that the best binding mode might not be the productive one, and the full reactions for a number of enzyme-substrate complexes have therefore to be considered to find the lowest-energy reaction pathway. Next, examples are given of how the cluster approach can help in the elucidation of detailed reaction mechanisms of biocatalytically interesting enzymes, and how this knowledge can be exploited to develop enzymes with new functions or to understand the reasons for lack of activity toward non-natural substrates. The enzymes discussed in this context are phenolic acid decarboxylase and metal-dependent decarboxylases from the amidohydrolase superfamily. Next, the application of the cluster approach in the investigation of enzymatic enantioselectivity is discussed. The reaction of strictosidine synthase is selected as a case study, where the cluster calculations could reproduce and rationalize the selectivities of both the natural and non-natural substrates. Finally, we discuss how the cluster approach can be used to guide the rational design of enzyme variants with improved activity and selectivity. Acyl transferase from Mycobacterium smegmatis serves as an instructive example here, for which the calculations could pinpoint the factors controlling the reaction specificity and enantioselectivity. The cases discussed in this Account highlight thus the value of the cluster approach as a tool in biocatalysis. It complements experiments and other computational techniques in this field and provides insights that can be used to understand existing enzymes and to develop new variants with tailored properties.

Differences in Formation Mechanisms of Phenolic Off-Flavor Compounds among Yeast Species

Phenolic off-flavor compounds such as 4-vinylguaiacol (4-VG) affect the quality of fermented foods. In Saccharomyces cerevisiae, 4-VG production requires prenylated FMN (flavin mononucleotide) produced by the mitochondrial protein Pad1. To examine this mechanism further, we expressed Escherichia coli ubiX, whose gene product prenylates FMN in E. coli, in an S. cerevisiae Δpad1 strain, which recovered the ability to produce 4-VG. Because E. coli UbiX is expected to localize to the cytoplasm in the transformed yeast, next, we expressed a truncated Pad1 (tPad1) variant lacking the mitochondrial translocation signal sequence in S. cerevisiae Δpad1. The transformed strain also recovered the ability to produce 4-VG; furthermore, it produced a larger amount of 4-VG than wild-type S. cerevisiae, indicating that 4-VG production in S. cerevisiae may be mediated by the partial mislocalization of Pad1 to the cytoplasm. We also expressed the gene encoding Dekkera (Brettanomyces) bruxellensis phenolic acid decarboxylase (DbPAD) in S. cerevisiae Δpad1. The transformed yeast recovered the ability to produce 4-VG, indicating that the D. bruxellensis phenolic acid decarboxylation reaction does not require prenylated FMN. Sequencing of the cloned DbPAD gene showed that the translation start codon differs from previous reports; therefore, we recloned the DbPAD gene from the position predicted to be the translation initiation point (designated DbPADs). Transformation of S. cerevisiae Δpad1 with DbPADs further increased the amount of 4-VG produced. Collectively, these findings provide insight into the mechanism of off-flavor production by different yeast species.

Genome analysis of Erwinia persicina reveals implications for soft rot pathogenicity in plants.

Soft rot disease causes devastating losses to crop plants all over the world, with up to 90% loss in tropical climates. To better understand this economically important disease, we isolated four soft rot-causing Erwinia persicina strains from rotted vegetables. Notably, E. persicina has only recently been identified as a soft rot pathogen and a comprehensive genomic analysis and comparison has yet to be conducted. Here, we provide the first genomic analysis of E. persicina, compared to Pectobacterium carotovorum, P. carotovorum, and associated Erwinia plant pathogens. We found that E. persicina shares common genomic features with other Erwinia species and P. carotovorum, while having its own unique characteristics as well. The E. persicina strains examined here lack Type II and Type III secretion systems, commonly used to secrete pectolytic enzymes and evade the host immune response, respectively. E. persicina contains fewer putative pectolytic enzymes than P. carotovorum and lacks the Out cluster of the Type II secretion system while harboring a siderophore that causes a unique pink pigmentation during soft rot infections. Interestingly, a putative phenolic acid decarboxylase is present in the E. persicina strains and some soft rot pathogens, but absent in other Erwinia species, thus potentially providing an important factor for soft rot. All four E. persicina isolates obtained here and many other E. persicina genomes contain plasmids larger than 100 kbp that encode proteins likely important for adaptation to plant hosts. This research provides new insights into the possible mechanisms of soft rot disease by E. persicina and potential targets for diagnostic tools and control measures.

Involvement of Cytochrome P450 in Organic-Solvent Tolerant Bacillus subtilis GRSW1-B1 in Vanillin Production via Ferulic Acid Metabolism

The detection of vanillin during the metabolism of ferulic acid by several Bacillus strains has been reported; however, its occurrence is not yet understood. Herein, the potential enzymes involved in vanillin production during ferulic acid metabolism in the previously reported butanol-tolerant Bacillus subtilis strain GRSW1-B1 were explored. The recombinant E. coli cells that overexpressed phenolic acid decarboxylase (PadC) rapidly converted ferulic acid to 4-vinylguaiacol. The detection of vanillin was concurrent with a decrease in 4-vinylguaiacol. In addition, the reversible abiotic conversion of 4-vinylguaiacol and apocynol was observed. The overexpression of CypD, a Bacillus P450, resulted in notable production of vanillin. The two-step conversion of ferulic acid yielded 145 μM over 72 h at pH 9. Vanillin yields of approximately 258 μM and 212 μM were obtained from ferulic acid metabolism by recombinant E. coli coexpressing PadC and CypD after conversion for 72 h, at pH 9 and 10, respectively. Several possibilities that underlie the production of vanillin were discussed. This information is useful for understanding ferulic acid metabolism by Bacillus strains and for further improving this strain as a host for the production of valuable compounds from biomass.

Multi-omics analysis of the metabolism of phenolic compounds in tea leaves by Aspergillus luchuensis during fermentation of pu-erh tea

Aspergillus fungi are extensively used in traditional food fermentation, so their functions, mechanisms, and safety risks are worth exploring. In this study, a dominant fungal strain (P1) was isolated from a fermented pu-erh tea and identified as A. luchuensis by phylogenetic analysis of fungal internally-transcribed spacer sequencing, partial β-tubulin and calmodulin genes. A pure-strain fermentation of tea leaves was developed, and tea compounds were analyzed by widely-targeted metabolomics, using high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). The mycotoxins, aflatoxin (B1, B2, M1 and M2), fumonisin B1 and B2, ochratoxin A, citrinin, were not detected in fermented tea leaves using methods in the National Standard of the Peoples’ Republic of China. The genome of 36.60 Mb with 11,836 protein-coding genes was sequenced by PacBio sequencing and annotated. Expression of fungal genes during fermentation was analyzed by Illumina HiSeq 2500; genes encoding enzymes including glycoside hydrolases, phenolic acid esterases, laccases, tyrosinases, dehydrogenases, peroxidases, dioxygenases, monooxygenases, decarboxylases and O-methyltransferases were identified. These enzymes catalyze hydrolysis, oxidation, ring cleavage, hydroxylation, decarboxylation and O-methylation of phenolic compounds , significantly (p < 0.05) changing the phenolic compound composition. While, phenolic compounds were degraded through degradation of aromatic compounds pathways and xenobiotics biodegradation and metabolism pathways. These findings advance knowledge of the functions and mechanisms of action of Aspergillus in traditional food fermentation.

Engineering and linker-mediated co-immobilization of carotenoid cleavage oxygenase with phenolic acid decarboxylase for efficiently converting ferulic acid into vanillin

The artificial cascade system with a coenzyme-independent decarboxylase and oxygenase was a very attractive approach for bioconversion of ferulic acid into vanillin. However, its potential was impeded by the low activity and thermostability of oxygenase. In this study, a combined strategy including the protein engineering of a carotenoid cleavage oxygenase from Thielavia terrestris (TtCCO) and its linker-mediated co-immobilization with a decarboxylase from Bacillus atrophaeus (BaPAD) was explored with aims to improve the catalytic efficiency and robustness of this artificial cascade system. We proved that engineering the enzyme by inserting an extra short peptide between E450 to E476 greatly affected TtCCO substrate specificity, catalytic activity, and thermostability. Among three variants, TtCCO2 displayed better thermostability and had 1.4-, 55- and 1.3-fold higher activity than TtCCO on 4-vinylguaiacol, resveratrol, and isoeugenol, respectively. The excellent protein and enzyme activity yield of immobilization was achieved by linker-mediated immobilization of 4lp-TtCCO2 @ mesoporous Y zeolite. A 100% conversion rate of ferulic acid and a 55% of the molar conversion yield of vanillin were obtained by co-immobilized 4lp-TtCCO2/4lp-BaPAD@mesoporous Y zeolite. This work demonstrated an effective engineering strategy for more efficient and robust oxygenases and the potential of this bi-enzyme cascade system for converting ferulic acid into vanillin.

A Magnetosome-Based Platform for Flow Biocatalysis.

Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein “cargo” can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average space–time yield of 49.2 mmol L–1 h–1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts.

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.

Phenolic acid decarboxylase of Aspergillus luchuensis plays a crucial role in 4-vinylguaiacol production during awamori brewing

Phenolic acid decarboxylase of <i>Aspergillus luchuensis</i> plays a crucial role in 4-vinylguaiacol production during <i>awamori</i> brewing