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

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. IMPORTANCE: 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.

Isolation and characterization of filamentous fungi capable of degrading the mycotoxin patulin.

Patulin is a toxic secondary metabolite synthesized by various fungal strains. This mycotoxin is generally toxic to microorganisms as well as mammals due to its reactivity with the important cellular antioxidant glutathione. In this study, we explored the presence of microorganisms capable of degrading patulin. Microorganisms were screened for the ability to both grow in culture medium containing patulin and reduce its concentration. Screening of 510 soil samples resulted in the isolation of two filamentous fungal strains, one of which, Acremonium sp. TUS-MM1 was characterized in detail. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that TUS-MM1 cells degraded patulin to desoxypatulinic acid. In addition, extracellular components of strain TUS-MM1 also exhibited patulin-transforming activity. High-performance liquid chromatography analysis revealed that the extracellular components generated several products from patulin. Disc diffusion assay using Escherichia coli cells revealed that the patulin-transformation products by the extracellular components are less toxic than patulin. We also demonstrated that a thermostable, low-molecular-weight compound within the extracellular components was responsible for the patulin-transforming activity. These results suggest that strain TUS-MM1 transforms patulin into less-toxic molecules by secreting a highly reactive compound. In addition, once patulin enters the cells, strain TUS-MM1 can transform it into desoxypatulinic acid to reduce its toxicity.

Planning and effectiveness of intensive rehabilitation as a treatment for a patient with neurosarcoidosis: A case report.

INTRODUCTION: Neurosarcoidosis tends to prolong the duration of treatment and may result in a decline in physical function requiring rehabilitation. Because of a rare disease, the adjustment of oral steroid dosage, which is the cornerstone of treatment, is highly dependent on professional experience in general. Therefore, the number of hospitals that can perform dosage adjustment is very limited, and it is difficult to provide concurrent intense rehabilitation at the same hospital over a long period of time, and there are no reports that mention this. PATIENT CONCERNS: A 49-year-old man, who presented with impaired consciousness, dysphagia and right hemiplegia, was diagnosed with neurosarcoidosis based on a previous diagnosis of sarcoidosis, laboratory test results, and clinical symptoms. High-dose oral steroid therapy was initiated and he was transferred to our rehabilitation hospital for progressive disuse approximately 2 months after the disease onset. DIAGNOSES: This case was diagnosed as "probable" neurosarcoidosis. INTERVENTIONS: The steroid dose was not reduced during rehabilitation treatment in our hospital considering the risk of relapse of the primary disease due to steroid reduction. His training regimen focused on minimum activities of daily living was performed, and its effectiveness was determined during approximately 60 days after the initiation of rehabilitation. OUTCOMES: Two months after admission, he was independently eating, transferring, and toileting under supervision. He was discharged home 3 months after admission. LESSONS: Intensive rehabilitation can be one of the effective comprehensive treatment strategy for patients with neurosarcoidosis. On the other hand, since there is no consensus treatment method, the duration of rehabilitation and goal setting should be adjusted based on an understanding of the characteristics of the disease and the overall treatment plan.

Diversity and characteristics of plant immunity-activating bacteria from Brassicaceae plants.

BACKGROUND: Microorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity-activating microorganisms in Brassicaceae plants. RESULTS: A total of 25 bacterial strains were isolated from the interior of a Brassicaceae plant, Raphanus sativus var. hortensis. Ten different genera of bacteria were identified: Pseudomonas, Leclercia, Enterobacter, Xanthomonas, Rhizobium, Agrobacterium, Pantoea, Rhodococcus, Microbacterium, and Plantibacter. The isolated strains were analyzed using a method to detect plant immunity-activating microorganisms that involves incubation of the microorganism with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses. In this method, cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells serves as a marker of immune activation. Among the 25 strains examined, 6 strains markedly enhanced cryptogein-induced ROS production in BY-2 cells. These 6 strains colonized the interior of Arabidopsis plants, and Pseudomonas sp. RS3R-1 and Rhodococcus sp. RS1R-6 selectively enhanced plant resistance to the bacterial pathogens Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovorum subsp. carotovorum NBRC 14082, respectively. In addition, Pseudomonas sp. RS1P-1 effectively enhanced resistance to both pathogens. We also comprehensively investigated the localization (i.e., cellular or extracellular) of the plant immunity-activating components produced by the bacteria derived from R. sativus var. hortensis and the components produced by previously isolated bacteria derived from another Brassicaceae plant species, Brassica rapa var. perviridis. Most gram-negative strains enhanced cryptogein-induced ROS production in BY-2 cells via the presence of cells themselves rather than via extracellular components, whereas many gram-positive strains enhanced ROS production via extracellular components. Comparative genomic analyses supported the hypothesis that the structure of lipopolysaccharides in the outer cell envelope plays an important role in the ROS-enhancing activity of gram-negative Pseudomonas strains. CONCLUSIONS: The assay method described here based on elicitor-induced ROS production in cultured plant cells enabled the discovery of novel plant immunity-activating bacteria from R. sativus var. hortensis. The results in this study also suggest that components involved in the ROS-enhancing activity of the bacteria may differ depending largely on genus and species.

Draft Genome Sequence of Acinetobacter calcoaceticus TUS-SO1, a Bacterium Capable of Degrading 2-Phenoxyacetophenone.

Acinetobacter calcoaceticus TUS-SO1 degrades 2-phenoxyacetophenone, a model compound for the ß-O-4 linkage in lignin. Here, we report the whole-genome sequence of this bacterium. The draft genome comprises 4,284,351 nucleotides and 3,976 coding DNA sequences, with an average G+C content of 38.5%.

Draft Genome Sequences of Endophytic Pseudomonas Strains, Isolated from the Interior of Brassicaceae Plants.

Members of the genus Pseudomonas have often been studied as agricultural biocontrol agents to activate plant immune responses. Here, we report the draft genome sequences of six Pseudomonas strains that were isolated from the interior of Brassicaceae plants.

Discovery of diphenyl ether-degrading Streptomyces strains by direct screening based on ether bond-cleaving activity.

Diphenyl ethers (DEs), which are widely used in the agricultural and chemical industries, have become hazardous contaminants in the environment. Although several DE-degrading bacteria have been reported, discovering new types of such microorganisms could enhance understanding of the degradation mechanism in the environment. In this study, we used a direct screening method based on detection of ether bond-cleaving activity to screen for microorganisms that degrade 4,4'-dihydroxydiphenyl ether (DHDE) as a model DE. Microorganisms isolated from soil samples were incubated with DHDE, and strains producing hydroquinone via ether bond cleavage were selected using hydroquinone-sensitive Rhodanine reagent. This screening procedure resulted in the isolation of 3 bacteria and 2 fungi that transform DHDE. Interestingly, all of the isolated bacteria belonged to one genus, Streptomyces. To our knowledge, these are the first microorganisms of the genus Streptomyces shown to degrade a DE. Streptomyces sp. TUS-ST3 exhibited high and stable DHDE-degrading activity. HPLC, LC-MS, and GC-MS analyses revealed that strain TUS-ST3 converts DHDE to its hydroxylated analogue and generates hydroquinone as an ether bond-cleavage product. Strain TUS-ST3 also transformed DEs other than DHDE. In addition, glucose-grown TUS-ST3 cells began to transform DHDE after incubation with this compound for 12 h, and produced 75 µM hydroquinone in 72 h. These activities of streptomycetes may play an important role in DE degradation in the environment. We also report the whole genome sequence of strain TUS-ST3.

Biocatalytic synthesis and evaluation of antioxidant and antibacterial activities of hydroxyequols.

Hydroxyequols are promising analogues of the biologically active flavonoid, equol. We recently found that the flavin-dependent monooxygenase HpaBro-3 of Rhodococcus opacus regioselectively synthesizes 3'-hydroxyequol from equol, whereas HpaBpl-1 of Photorhabdus luminescens synthesizes 6-hydroxyequol. In this study, we investigated the cascade synthesis of a dihydroxyequol compound from equol using these two enzymes. When Escherichia coli cells expressing HpaBro-3 and cells expressing HpaBpl-1 were simultaneously incubated with equol, the cells efficiently synthesized 6,3'-dihydroxyequol (8.7 mM, 2.4 g/L) via 3'- and 6-hydroxyequols in one pot. The antioxidant activity of the equol derivatives increased with an increase in the number of hydroxyl groups on the equol scaffold. 6,3'-Dihydroxyequol exhibited potent antioxidant activity. In addition, 6-hydroxyequol significantly inhibited the growth of E. coli. Cell survival studies suggested that 6-hydroxyequol is a bactericidal rather than bacteriostatic compound. To our knowledge, this is the first report describing the antibacterial activity of hydroxyequols.

Sustainable Approach for Peroxygenase-Catalyzed Oxidation Reactions Using Hydrogen Peroxide Generated from Spent Coffee Grounds and Tea Leaf Residues.

Peroxygenases are promising catalysts for use in the oxidation of chemicals as they catalyze the direct oxidation of a variety of compounds under ambient conditions using hydrogen peroxide (H2O2) as an oxidant. Although the use of peroxygenases provides a simple method for oxidation of chemicals, the anthraquinone process currently used to produce H2O2 requires significant energy input and generates considerable waste, which negatively affects process sustainability and production costs. Thus, generating H2O2 for peroxygenases on site using an environmentally benign method would be advantageous. Here, we utilized spent coffee grounds (SCGs) and tea leaf residues (TLRs) for the production of H2O2. These waste biomass products reacted with molecular oxygen and effectively generated H2O2 in sodium phosphate buffer. The resulting H2O2 was utilized by the bacterial P450 peroxygenase, CYP152A1. Both SCG-derived and TLR-derived H2O2 promoted the CYP152A1-catalyzed oxidation of 4-methoxy-1-naphthol to Russig's blue as a model reaction. In addition, when CYP152A1 was incubated with styrene, the SCG and TLR solutions enabled the synthesis of styrene oxide and phenylacetaldehyde. This new approach using waste biomass provides a simple, cost-effective, and sustainable oxidation method that should be readily applicable to other peroxygenases for the synthesis of a variety of valuable chemicals.

Isolation and characterization of microorganisms capable of cleaving the ether bond of 2-phenoxyacetophenone.

Lignin is a heterogeneous aromatic polymer and major component of plant cell walls. The ß-O-4 alkyl aryl ether is the most abundant linkage within lignin. Given that lignin is effectively degraded on earth, as yet unknown ether bond-cleaving microorganisms could still exist in nature. In this study, we searched for microorganisms that transform 2-phenoxyacetophenone (2-PAP), a model compound for the ß-O-4 linkage in lignin, by monitoring ether bond cleavage. We first isolated microorganisms that grew on medium including humic acid (soil-derived organic compound) as a carbon source. The isolated microorganisms were subsequently subjected to colorimetric assay for 2-PAP ether bond-cleaving activity; cells of the isolated strains were incubated with 2-PAP, and strains producing phenol via ether bond cleavage were selected using phenol-sensitive Gibbs reagent. This screening procedure enabled the isolation of various 2-PAP-transforming microorganisms, including 7 bacteria (genera: Acinetobacter, Cupriavidus, Nocardioides, or Streptomyces) and 1 fungus (genus: Penicillium). To our knowledge, these are the first microorganisms demonstrated to cleave the ether bond of 2-PAP. One Gram-negative bacterium, Acinetobacter sp. TUS-SO1, was characterized in detail. HPLC and GC-MS analyses revealed that strain TUS-SO1 oxidatively and selectively cleaves the ether bond of 2-PAP to produce phenol and benzoate. These results indicate that the transformation mechanism differs from that involved in reductive ß-etherase, which has been well studied. Furthermore, strain TUS-SO1 efficiently transformed 2-PAP; glucose-grown TUS-SO1 cells converted 1 mM 2-PAP within only 12 h. These microorganisms might play important roles in the degradation of lignin-related compounds in nature.

Diversity and characteristics of culturable endophytic bacteria from Passiflora edulis seeds.

Defense compounds generally inhibit microbial colonization of plants. In this study, we examined the presence of endophytes in Passiflora edulis seeds that accumulate resveratrol and piceatannol at extremely high levels as defense compounds. Interestingly, although no microbial colonies appeared on an agar growth medium from the cut or homogenized seeds, colonies were generated from cut seedlings derived from the seeds. A total of 19 bacterial strains were isolated, of which 15 were classified as Gram-positive. As we hypothesized that extremely high levels of piceatannol in the seeds would inhibit the growth of endophytes cultured directly from the seeds, we examined the antimicrobial activity of this compound against the isolated bacteria. Piceatannol exerted bacteriostatic rather than bactericidal effects on most of the bacteria tested. These results suggest that the bacteria remain static in the seeds due to the presence of piceatannol and are transmitted to the seedlings during the germination process, enabling colonies to be established from the seedlings on the agar medium. We also investigated the biocatalytic activity of the isolated bacteria toward resveratrol and piceatannol. One bacterium, Brevibacterium sp. PE28-2, converted resveratrol and piceatannol to their respective derivatives. This strain is the first endophyte shown to exhibit such activity.

An efficient direct screening system for microorganisms that activate plant immune responses based on plant-microbe interactions using cultured plant cells.

Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant-microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell-cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.

Efficient and long-term vanillin production from 4-vinylguaiacol using immobilized whole cells expressing Cso2 protein.

Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E. coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E. coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E. coli cells for both continuous and repeated batch vanillin production without any coenzymes.

Isolation and characterization of Gram-negative and Gram-positive bacteria capable of producing piceatannol from resveratrol.

Piceatannol is a valuable natural polyphenol with therapeutic potential in cardiovascular and metabolic disease treatment. In this study, we screened for microorganisms capable of producing piceatannol from resveratrol via regioselective hydroxylation. In the first screening, we isolated microorganisms utilizing resveratrol, phenol, or 4-hydroxyphenylacetic acid as a carbon source for growth. In the second screening, we assayed the isolated microorganisms for hydroxylation of resveratrol. Using this screening procedure, a variety of resveratrol-converting microorganisms were obtained. One Gram-negative bacterium, Ensifer sp. KSH1, and one Gram-positive bacterium, Arthrobacter sp. KSH3, utilized 4-hydroxyphenylacetic acid as a carbon source for growth and efficiently hydroxylated resveratrol to piceatannol without producing any detectable by-products. The hydroxylation activity of strains KSH1 and KSH3 was strongly induced by cultivation with 4-hydroxyphenylacetic acid as a carbon source during stationary growth phase. Using the 4-hydroxyphenylacetic acid-induced cells as a biocatalyst under optimal conditions, production of piceatannol by strains KSH1 and KSH3 reached 3.6 mM (0.88 g/L) and 2.6 mM (0.64 g/L), respectively. We also cloned genes homologous to the monooxygenase gene hpaBC from strains KSH1 and KSH3. Introduction of either hpaBC homolog into Escherichia coli endowed the host with resveratrol-hydroxylating activity.

Monooxygenase-catalyzed regioselective hydroxylation for the synthesis of hydroxyequols.

Monooxygenases exhibiting high activity and differing regioselectivity for the dietary isoflavone metabolite equol were discovered among enzymes in the HpaBC family by a genome mining approach. These enzymes enabled the one-step product-selective synthesis of 3'- and 6-hydroxyequols from equol and molecular oxygen.

Efficient monooxygenase-catalyzed piceatannol production: Application of cyclodextrins for reducing product inhibition.

Piceatannol is a rare, costly plant-based stilbene derivative and exhibits various health-enhancing properties. Recently, we demonstrated that piceatannol could be produced from resveratrol through site-selective hydroxylation using Escherichia coli cells expressing the monooxygenase HpaBC. However, piceatannol production ceased at approximately 25 mM, even when sufficient levels of the substrate resveratrol remained in the reaction mixture. In this study, we found that high concentrations (>20-25 mM) of piceatannol significantly inhibited the HpaBC-catalyzed reaction. Cyclodextrins (CDs) reportedly encapsulate various hydrophobic compounds. We found that the addition of ß-CD or γ-CD to the reaction mixture reduced the inhibition caused by the product piceatannol. The effects of ß-CD on piceatannol production were more pronounced than those of γ-CD at high concentrations of the substrate resveratrol and CDs. The production of piceatannol reached 49 mM (12 g L-1) in the presence of ß-CD, a level twice that achieved in the absence of ß-CD. The technique described here might be applicable to the bioproduction of other stilbenes and structurally related compounds.

Biotechnological production of vanillin using immobilized enzymes.

Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sepabeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8mg yield of vanillin from isoeugenol through ten reaction cycles at a 1mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production.

Biocatalytic synthesis of 3,4,5,3',5'-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC.

HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3',5'-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100 mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9 mM (1.8 g L(-1)) in 48 h.

Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism.

Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol.

High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process.

Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L(-1)) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.