A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Purification,characterization and gene cloning of isoeugenol-degrading enzyme from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27

An isoeugenol-degrading enzyme was purified to homogeneity from Pseudomonas putida IE27, an isoeugenol-assimilating bacterium. The purified enzyme was a 55 kDa monomer and catalyzed the initial step of isoeugenol degradation, the oxidative cleavage of the side chain double-bond of isoeugenol, to form vanillin. Another reaction product of isoeugenol degradation besides vanillin was identified to be acetaldehyde. The values of Km and k _cat for isoeugenol were 175 μM and 5.18 s^–1, respectively. The purified enzyme catalyzed the incorporation of an oxygen atom from either molecular oxygen or water into vanillin, suggesting that the isoeugenol-degrading enzyme is a kind of monooxygenase. The gene encoding the isoeugenol-degrading enzyme and its flanking regions were isolated from P. putida IE27. The amino acid sequence of the enzyme was similar to those of lignostilbene-α,β-dioxygenases, carotenoid monooxygenases and 9-cis-epoxycarotenoid dioxygenases.     

Biotransformation of Isoeugenol to Vanillin by a Novel Strain of <Emphasis Type="Italic">Bacillus fusiformis</Emphasis>

A novel strain of Bacillus fusiformis, producing high amounts of vanillin from isoeugenol, was isolated from soil. Using 60% (v/v) isoeugenol as substrate and solvent and at pH 4.0, 37 °C and 180 rpm, vanillin was produced at 32.5 g l⁻¹ over 72 h. The unused isoeugenol was reusable.     

Vanillin production using <Emphasis Type="Italic">Escherichia coli</Emphasis> cells over-expressing isoeugenol monooxygenase of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.     

NMR-based metabolomic analysis of the seasonal and regional variability of phytochemical compounds in Curtisia dentata stem bark

Traditional medicine markets are provided with medicinal plant material throughout the year, however, internal (e.g. plant age, genetic variability and differential expression of genes) and external factors (e.g. water and nutrient availability, rainfall, photoperiod and herbivory), affect secondary metabolite production in plants. In this study, seasonal variability in metabolite production in Curtisia dentata trees from two geographically separated regions in South Africa are compared. NMR analysis of C. dentata stem bark samples yielded spectral data which were processed in MestReNova to perform multivariate data analysis using Soft Independent Modeling of Class Analogy (SIMCA) software. This study shows that there are not only seasonal, regional and yearly differences in secondary metabolite production in C. dentata trees, but that production patterns of hydrophilic and lipophilic chemical compounds in individual trees also vary. Sucrose, isoeugenol and betulinic acid have been used in a targeted analysis to show the variation in individual compounds in the individual trees as a response to seasonal and geographical differences. Therefore, the season and year, as well as the region, harvesting site and specific trees from which plant material is collected affect the concentrations of chemical compounds extracted from C. dentata stem bark for the preparation of remedies.     

转化异丁香酚生成香草醛纺锤芽孢杆菌的筛选

以底物异丁香酚为唯一碳源从七壤中筛选获得了一株能高效转化异丁香酚生成香草醛的芽孢杆菌。根据生理生化特性及16SrRNA序列分析鉴定其属于纺锤芽孢杆菌（Bacillus fusiformis）,初步试验表明该菌能转化2％异丁香酚生成4．20g/L香草醚。     

Metabolic engineering in strawberry fruit uncovers a dormant biosynthetic pathway

Wild strawberry (Fragaria vesca) fruit contains several important phenylpropene aroma compounds such as eugenol, but cultivated varieties are mostly devoid of them. We have redirected the carbon flux in cultivated strawberry (Fragaria×ananassa) fruit from anthocyanin pigment biosynthesis to the production of acetates of hydroxycinnamyl alcohols, which serve as the precursors of the phenylpropenes, by downregulating the strawberry chalcone synthase (CHS) via RNAi-mediated gene silencing and, alternatively, by an antisense CHS construct. Simultaneous heterologous overexpression of a eugenol (EGS) and isoeugenol synthase (IGS) gene in the same cultivated strawberry fruits boosted the formation of eugenol, isoeugenol, and the related phenylpropenes chavicol and anol to concentrations orders of magnitude greater than their odor thresholds. The results show that Fragaria×ananassa still bears a phenylpropene biosynthetic pathway but the carbon flux is primarily directed to the formation of pigments. Thus, partial restoration of wild strawberry flavor in cultivated varieties is feasible by diverting the flavonoid pathway to phenylpropene synthesis through metabolic engineering.     

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10 g l⁻¹ isoeugenol was converted to 3.75 g l⁻¹ vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography–mass spectrometry. Based on the accurate masses obtained from gas chromatography–high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.     

Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.