Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1). This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1). The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

Candida galli Strain PGO6: A Novel Isolated Yeast Strain Capable of Transformation of Isoeugenol into Vanillin and Vanillic Acid

Candida galli strain PGO6 isolated from oil-contaminated water is the first isolated yeast strain which is capable to form vanillin and vanillic acid during isoeugenol biotransformation. The products were confirmed by thin-layer chromatography (TLC), changes in the UV absorption pattern and high-performance liquid chromatography (HPLC). The phenotypic and physiochemical characteristics as well as molecular phylogenetic analysis based on amplification the ITS1-5.8S-ITS2 rDNA regions indicated the isolated strain PGO6 was identified as C. galli (GenBank accession number HM641231). Resting cells of C. galli PGO6 from the late-exponential of growth phase were used as biocatalysts for the biotransformation of isoeugenol. The optimal molar conversion of vanillin (48%) and vanillic acid (19%) was obtained after a 30 h incubation using 0.1% (v/v) of isoeugenol and 6 mg of dry weight of cells per ml without further optimization. Under these conditions, the total amount of vanillin and vanillic acid was 583 mg l(-1). Further biotransformation was carried out using 0.5% (v/v) of isoeugenol under the resting cells conditions, yielding a vanillin concentration of 1.12 g l(-1) (molar yield 25.7%) after 60 h incubation. This study brings the first evidence for biotransformation of isoeugenol to vanillin and vanillic acid by a yeast strain.     

Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167

To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram-positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected.     

Biotransformation of eugenol to vanillin by a novel strain Bacillus safensis SMS1003

Due to the extensive applications of vanillin as flavored compound and increasing consumers concern for its natural and environment friendly mode of production, present work was focused on the selection of bacterial isolate capable of producing vanillin using eugenol biotransformation. Bacterial strain SMS1003 is evidenced as the potential strain for vanillin production and identified as Bacillus safensis (GeneBank accession no. MG561863) using biochemical tests and molecular phylogenic analysis of its 16S rDNA gene sequence. Molar yield of vanillin reached up to 10.7% (0.055?g/L) at 96?h of biotransformation using growing culture of B. safensis SMS1003 in following culture conditions: eugenol concentration 500?mg/L; temperature 37?°C; initial pH 7.0; inoculum volume 4%; volume of culture media 10%; and shaking speed 180?rpm. Vanillin was detected as the single metabolite with a molar yield of 26% (0.12?g/L) at 96?h using resting cells of B. safensis SMS1003. Product confirmation was based on spectral scan using photodiode array detector, Fourier-transform infrared spectroscopy, high-performance liquid chromatography, and mass spectroscopy.     

Characterization of the eugenol hydroxylase genes (ehyA/ehyB) from the new eugenol-degrading Pseudomonas sp. strain OPS1

During the screening for bacteria capable of converting eugenol to vanillin, strain OPS1 was isolated, which was identified as a new Pseudomonas species by 16 s rDNA sequence analysis. When this bacterium was grown on eugenol, the intermediates, coniferyl alcohol, ferulic acid, vanillic acid, and protocatechuic acid, were identified in the culture supernatant. The genes encoding the eugenol hydroxylase (ehyA, ehyB), which catalyzes the first step of this biotransformation, were identified in a genomic library of Pseudomonas sp. strain OPS1 by complementation of the eugenol-negative mutant SK6165 of Pseudomonas sp. strain HR199. EhyA and EhyB exhibited 57% and 85% amino acid identity to the eugenol hydroxylase subunits of Pseudomonas sp. strain HR199 and up to 34% and 54% identity to the corresponding subunits of p-cresol methylhydroxylase from P. putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for an eugenol dehydrogenase of P fluorescens E118 corresponded well with the appropriate regions of EhyA and EhyB. Downstream of ehyB, an open reading frame was identified, whose deduced amino acid sequence exhibited up to 71% identity to azurins, representing most probably the gene (azu) of the physiological electron acceptor of the eugenol hydroxylase. The eugenol hydroxylase genes were amplified by PCR, cloned, and functionally expressed in Escherichia coli.     

Highly Efficient Biotransformation of Eugenol to Ferulic Acid and Further Conversion to Vanillin in Recombinant Strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter⁻¹. This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter⁻¹ after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter⁻¹, besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter⁻¹. The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

微生物转化方法生产香草酸与香草醛的初步研究

从实验室保藏的菌种中筛选到一株黑曲霉（Aspergillus niger）SW-33,能够将1g/L的阿魏酸底物转化为0.23g/L的香草酸,相应的摩尔转化率为29.35％;流加四次底物阿魏酸后,产物浓度达到1.11g/L,相应的摩尔转化率为44.9％。为了提高产物浓度,对培养基和发酵条件进行优化,使得该菌株能够将1g/l的阿魏酸底物转化为0.46g/L的香草酸,相应的摩尔转化率为57.81％。提取得到的香草酸,经HPLC测定,纯度为85.9％;提取收率为75.2％。用含香草酸的转化液,或者用提取的结晶香草酸,加入朱红密孔菌（Prcnporus cinnabarnus）SW-0203发酵培养液,可得到转化产物香草醛。     

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.     

Biotransformation of eugenol to ferulic acid by a recombinant strain of Ralstonia eutropha H16

The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.     

黑曲霉CGMCC0774和朱红密孔菌CGMCC1115两步转化阿魏酸制备生物香兰素

在25 L发酵罐中黑曲霉Aspergillus niger CGMCC0774转化阿魏酸可生成香草酸2.24 g/L,摩尔转化率64.6%;朱红密孔菌Pycnoporus cinnabarinus CGMCC1115转化提取的香草酸可生成香草醛1.45 g/L,摩尔转化率为79.9%。将两步微生物转化有机串联,即用黑曲霉转化液加预先培养的朱红密孔菌Pycnoporus cinnabarinus CGMCC1115菌丝体继续转化,可产香草醛1.06 g/L,对原料阿魏酸的摩尔转化率34.0%。用米糠提取的天然阿魏酸做原料,两步串联微生物转化制备的生物香兰素经13C同位素的分析,符合生物香草素的等同要求。     

Biotransformation of Eugenol to Ferulic Acid by a Recombinant Strain of Ralstonia eutropha H16

The gene loci ehyAB, calA, and calB, encoding eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase, respectively, which are involved in the first steps of eugenol catabolism in Pseudomonas sp. strain HR199, were amplified by PCR and combined to construct a catabolic gene cassette. This gene cassette was cloned in the newly designed broad-host-range vector pBBR1-JO2 (pBBR1-JO2ehyABcalAcalB) and transferred to Ralstonia eutropha H16. A recombinant strain of R. eutropha H16 harboring this plasmid expressed functionally active eugenol hydroxylase, coniferyl alcohol dehydrogenase, and coniferyl aldehyde dehydrogenase. Cells of R. eutropha H16(pBBR1-JO2ehyABcalAcalB) from the late-exponential growth phase were used as biocatalysts for the biotransformation of eugenol to ferulic acid. A maximum conversion rate of 2.9 mmol of eugenol per h per liter of culture was achieved with a yield of 93.8 mol% of ferulic acid from eugenol within 20 h, without further optimization.     

Towards a high-yield bioconversion of ferulic acid to vanillin

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l⁻¹. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l⁻¹ could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l⁻¹ and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l⁻¹ were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes. Received: 15 October 1998 / Received revision: 13 January 1999 / Accepted: 18 January 1999     

Production of methoxyphenol-type natural aroma chemicals by biotransformation of eugenol with a new Pseudomonas sp.

In our screening program for microorganisms that are able to metabolize eugenol, the main component of the essential oil of the clove tree Syzigium aromaticum (sy. Eugenia cariophyllus), we found a new Pseudomonas sp. that produces several substituted methoxyphenols when eugenol is fed to the culture. A taxonomic characterization of this new organism has been performed. Examples of the biotransformation products, produced in high amounts, were vanillic acid with 3.25 g/l within 99 h, ferulic acid with 5.8 g/l within 75 h and coniferyl alcohol with 3.22 g/l within 47.5 h. By changing the culture conditions the ratio of the different metabolites could be varied. Based on these results a scheme for the degradation of eugenol by this strain has been established. Received: 1 April 1996 / Received revision: 24 June 1996 / Accepted: 1 July 1996     

Production of natural value-added compounds: an insight into the eugenol biotransformation pathway

During the past few years, the production of natural value-added compounds from microbial sources has gained tremendous importance. Due to an increase in consumer demand for natural products, various food and pharmaceutical industries are continuously in search of novel metabolites obtained from microbial biotransformation. The exploitation of microbial biosynthetic pathways is both feasible and cost effective in the production of natural compounds. The environmentally compatible nature of these products is one major reason for their increasing demand. Novel approaches for natural product biogeneration will take advantage of the current studies on biotechnology, biochemical pathways and microbiology. The interest of the scientific community has shifted toward the use of microbial bioconversion for the production of valuable compounds from natural substrates. The present review focuses on eugenol biotransformation by microorganisms resulting in the formation of various value-added products such as ferulic acid, coniferyl alcohol, vanillin and vanillic acid.     

Enhanced vanillin production from ferulic acid using adsorbent resin

High vanillin productivity was achieved in the batch biotransformation of ferulic acid by Streptomyces sp. strain V-1. Due to the toxicity of vanillin and the product inhibition, fed-batch biotransformation with high concentration of ferulic acid was unsuccessful. To solve this problem and improve the vanillin yield, a biotransformation strategy using adsorbent resin was investigated. Several macroporous adsorbent resins were chosen to adsorb vanillin in situ during the bioconversion. Resin DM11 was found to be the best, which adsorbed the most vanillin and the least ferulic acid. When 8% resin DM11 (wet w/v) was added to the biotransformation system, 45 g l⁻¹ ferulic acid could be added continually and 19.2 g l⁻¹ vanillin was obtained within 55 h, which was the highest vanillin yield by bioconversion until now. This yield was remarkable for exceeding the crystallization concentration of vanillin and therefore had far-reaching consequence in its downstream processing.     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Rapid degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated strain of bacillus coagulans

A new strain Bacillus coagulans BK07 was isolated from decomposed wood-bark, based on its ability to grow on ferulic acid as a sole carbon source. This strain rapidly decarboxylated ferulic acid to 4-vinylguaiacol, which was immediately converted to vanillin and then oxidized to vanillic acid. Vanillic acid was further demethylated to protocatechuic acid. Above 95% substrate degradation was obtained within 7 h of growth on ferulic acid medium, which is the shortest period of time reported to date. The major degradation products, was isolated and identified by thin-layer chromatography, high performance liquid chromatography and ¹H-nuclear magnetic resonance spectroscopy were 4-vinylguaiacol, vanillin, vanillic acid and protocatechuic acid.     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Biotransformation of isoeugenol to vanillin by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> IE27 cells

The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 °C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.     

Microbial transformation of ferulic acid to vanillic acid by <Emphasis Type="Italic">Streptomyces sannanensis</Emphasis> MTCC 6637

Streptomyces sannanensis MTCC 6637 was examined for its potentiality to transform ferulic acid into its corresponding hydroxybenzoate-derivatives. Cultures of S. sannanensis when grown on minimal medium containing ferulic acid as sole carbon source, vanillic acid accumulation was observed in the medium as the major biotransformed product along with transient formation of vanillin. A maximum amount of 400 mg/l vanillic acid accumulation was observed, when cultures were grown on 5 mM ferulic acid at 28°C. This accumulation of vanillic acid was found to be stable in the culture media for a long period of time, thus facilitating its recovery. Purification of vanillic acid was achieved by gel filtration chromatography using Sephadex™ LH-20 matrix. Catabolic route of ferulic acid biotransformation by S. sannanensis has also been demonstrated. The metabolic inhibitor experiment [by supplementation of 3,4 methylenedioxy-cinnamic acid (MDCA), a metabolic inhibitor of phenylpropanoid enzyme 4-hydroxycinnamoyl-CoA ligase (4-CL) along with ferulic acid] suggested that biotransformation of ferulic acid into vanillic acid mainly proceeds via CoA-dependent route. In vitro conversions of ferulic acid to vanillin, vanillic acid and vanillin to vanillic acid were also demonstrated with cell extract of S. sannanensis. Further degradation of vanillic acid to other intermediates such as, protocatechuic acid and guaiacol was not observed, which was also confirmed in vitro with cell extract.