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     

Vanillin production from simple phenols by wine-associated lactic acid bacteria

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.     

Microencapsulated bacterial cells can be used to produce the enzyme feruloyl esterase: preparation and in-vitro analysis

Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-l-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.     

Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid

Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficiant to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86 % within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.     

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。     

Studies on the Flavors in Saké

A very small amount of vanillin was found in Saké, but the mechanism of its formation during Saké brewing has not yet been elucidated. Therefore, shaking culture of a Saké yeast (Kyokai No. 7 strain) was carried out in the Hayduck’s solution containing ferulic acid which was considered to be a precursor of vanillin. By the analysis of the fermentation products, formation of p-hydroxybenzoic acid and vanillic acid was elucidated. On the other hand, in the similar experiment using vanillin in place of ferulic acid, p-hydroxybenzoic acid, p-hydroxybenzaldehyde and vanillic acid were identified.

On these results, it was suggested that vanillin might be formed as an intermediate of the degradation reaction of ferulic acid, and also, the demethoxylation of vanillin might be occurred in the fermentation of yeast.     

A complete enzymatic recovery of ferulic acid from corn residues with extracellular enzymes from Neosartorya spinosa NRRL185

An economic ferulic acid recovery from biomass via biological methods is of interest for a number of reasons. Ferulic acid is a precursor to vanillin synthesis. It is also a known antioxidant with potential food and medical applications. Despite its universal presence in all plant cell wall material, the complex structure of the plant cell wall makes ferulic acid recovery from biomass a challenging bioprocess. Previously, without pretreatment, very low (3-13%) recovery of ferulic acid from corn residues was achieved. We report here the discovery of a filamentous fungus Neosartorya spinosa NRRL185 capable of producing a full complement of enzymes to release ferulic acid and the development of an enzymatic process for a complete recovery of ferulic acid from corn bran and corn fibers. A partial characterization of the extracellular proteome of the microbe revealed the presence of at least seven cellulases and hemicellulases activities, including multiple iso-forms of xylanase and ferulic acid esterase. The recovered ferulic acid was bio-converted to vanillin, demonstrating its potential application in natural vanillin synthesis. The enzymatic ferulic acid recovery accompanied a significant release of reducing sugars (76-100%), suggesting much broader applications of the enzymes and enzyme mixtures from this organism.     

Enhanced vanillin production from recombinant E. coli using NTG mutagenesis and adsorbent resin

Vanillin production was tested with different concentrations of added ferulic acid in E. coli harboring plasmid pTAHEF containing fcs (feruloyl-CoA synthase) and ech (enoyl-CoA hydratase/aldolase) genes cloned from Amycolatopsis sp. strain HR104. The maximum production of vanillin from E. coli DH5alpha harboring pTAHEF was found to be 1.0 g/L at 2.0 g/L of ferulic acid for 48 h of culture. To improve the vanillin production by reducing its toxicity, two approaches were followed: (1) generation of vanillin-resistant mutant of NTG-VR1 through NTG mutagenesis and (2) removal of toxic vanillin from the medium by XAD-2 resin absorption. The vanillin production of NTG-VR1 increased to three times at 5 g/L of ferulic acid when compared with its wild-type strain. When 50% (w/v) of XAD-2 resin was employed in culture with 10 g/L of ferulic acid, the vanillin production of NTG-VR1 was 2.9 g/L, which was 2-fold higher than that obtained with no use of the resin.     

Production of vanillin from waste residue of rice bran oil by Aspergillus niger and Pycnoporus cinnabarinus

A new technology of transforming ferulic acid, which was from waste residue of rice bran oil, into vanillin was developed by a combination of fungal strains Aspergillus niger CGMCC0774 and Pycnoporus cinnabarinus CGMCC1115. Various concentrations of ferulic acid were compared, and the highest yield reached 2.2 g l(-1) of vanillic acid by A. niger CGMCC0774 in a 25 l fermenter when concentration of ferulic acid was 4 g l(-1). The filtrate of A. niger CGMCC0774 culture was concentrated and vanillic acid in the filtrate was bio-converted into vanillin by P. cinnabarinus CGMCC1115. The yield of vanillin reached 2.8 g l(-1) when 5 g l(-1) of glucose and 25 g of HZ802 resin were supplemented in the bioconversion medium. The 13C isotope analysis indicated that delta13C(PDB) of vanillin prepared was much different from chemically synthesized vanillin.     

Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid

Vanillin is one of the most important flavors in the food industry and there is great interest in its production through biotechnological processes starting from natural substrates such as ferulic acid. Among bacteria, recombinant Escherichia coli strains are the most efficient vanillin producers, whereas Pseudomonas spp. strains, although possessing a broader metabolic versatility, rapidly metabolize various phenolic compounds including vanillin. In order to develop a robust Pseudomonas strain that can produce vanillin in high yields and at high productivity, the vanillin dehydrogenase (vdh)-encoding gene of Pseudomonas fluorescens BF13 strain was inactivated via targeted mutagenesis. The results demonstrated that engineered derivatives of strain BF13 accumulate vanillin if inactivation of vdh is associated with concurrent expression of structural genes for feruloyl-CoA synthetase (fcs) and hydratase/aldolase (ech) from a low-copy plasmid. The conversion of ferulic acid to vanillin was enhanced by optimization of growth conditions, growth phase and parameters of the bioconversion process. The developed strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin produced by a Pseudomonas strain to date and opens new perspectives in the use of bacterial biocatalysts for biotechnological production of vanillin from agro-industrial wastes which contain ferulic acid.     

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.     

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.     

Lactic acid bacteria as a tool for biovanillin production: A review

Vanilla is the most commonly used natural flavoring agent in industries like food, flavoring, medicine, and fragrance. Vanillin can be obtained naturally, chemically, or through a biotechnological process. However, the yield from vanilla pods is low and does not meet market demand, and the use of vanillin produced by chemical synthesis is restricted in the food and pharmaceutical industries. As a result, the biotechnological process is the most efficient and cost-effective method for producing vanillin with consumer-demanding properties while also supporting industrial applications. Toxin-free biovanillin production, based on renewable sources such as industrial wastes or by-products, is a promising approach. In addition, only natural-labeled vanillin is approved for use in the food industry. Accordingly, this review focuses on biovanillin production from lactic acid bacteria (LAB), which is generally recognized as safe (GRAS), and the cost-cutting efforts that are utilized to improve the efficiency of biotransformation of inexpensive and readily available sources. LABs can utilize agro-wastes rich in ferulic acid to produce ferulic acid, which is then employed in vanillin production via fermentation, and various efforts have been applied to enhance the vanillin titer. However, different designs, such as response surface methods, using immobilized cells or pure enzymes for the spontaneous release of vanillin, are strongly advised.     

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.     

Bioengineering of the Plant Culture of <Emphasis Type="Italic">Capsicum frutescens</Emphasis> with Vanillin Synthase Gene for the Production of Vanillin

Production of vanillin by bioengineering has gained popularity due to consumer demand toward vanillin produced by biological systems. Natural vanillin from vanilla beans is very expensive to produce compared to its synthetic counterpart. Current bioengineering works mainly involve microbial biotechnology. Therefore, alternative means to the current approaches are constantly being explored. This work describes the use of vanillin synthase (VpVAN), to bioconvert ferulic acid to vanillin in a plant system. The VpVAN enzyme had been shown to directly convert ferulic acid and its glucoside into vanillin and its glucoside, respectively. As the ferulic acid precursor and vanillin were found to be the intermediates in the phenylpropanoid biosynthetic pathway of Capsicum species, this work serves as a proof-of-concept for vanillin production using Capsicum frutescens (C. frutescens or hot chili pepper). The cells of C. frutescens were genetically transformed with a codon optimized VpVAN gene via biolistics. Transformed explants were selected and regenerated into callus. Successful integration of the gene cassette into the plant genome was confirmed by polymerase chain reaction. High-performance liquid chromatography was used to quantify the phenolic compounds detected in the callus tissues. The vanillin content of transformed calli was 0.057% compared to 0.0003% in untransformed calli.     

Directing vanillin production from ferulic acid by increased acetyl-CoA consumption in recombinant Escherichia coli

The amplification of gltA gene encoding citrate synthase of TCA cycle was required for the efficient conversion of acetyl-CoA, generated during vanillin production from ferulic acid, to CoA, which is essential for vanillin production. Vanillin of 1.98 g/L was produced from the E. coli DH5alpha (pTAHEF-gltA) with gltA amplification in 48 h of culture at 3.0 g/L of ferulic acid, which was about twofold higher than the vanillin production of 0.91 g/L obtained by the E. coli DH5alpha (pTAHEF) without gltA amplification. The icdA gene encoding isocitrate dehydrogenase of TCA cycle was deleted to make the vanillin producing E. coli utilize glyoxylate bypass which enables more efficient conversion of acetyl-CoA to CoA in comparison with TCA cycle. The production of vanillin by the icdA null mutant of E. coli BW25113 harboring pTAHEF was enhanced by 2.6 times. The gltA amplification of the glyoxylate bypass in the icdA null mutant remarkably increased the production rate of vanillin with a little increase in the amount of vanillin production. The real synergistic effect of gltA amplification and icdA deletion was observed with use of XAD-2 resin reducing the toxicity of vanillin produced during culture. Vanillin of 5.14 g/L was produced in 24 h of the culture with molar conversion yield of 86.6%, which is the highest so far in vanillin production from ferulic acid using recombinant E. coli.     

Dissimilation of ferulic acid byBacillus subtilis

Bacillus subtilis utilized ferulic acid and its intermediates vanillin, vanillic acid, and protocatechuic acid as sole carbon source. The enzymes of the ferulic acid degradative pathway such as deacetylase, vanillin oxidase, vanillate-o-demethylase, and protocatechuate 3,4-dioxygenase were inducible in nature. Concentration of the inducer profoundly influenced the induction of the enzymes involved in ferulic acid dissimilation.     

Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5-7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.     

Vanilla Flavour Production Through Biotransformation Using Capsicum frutescens Root Cultures

Normal roots of Capsicum frutescens were excised from tissue-cultured plants into half strength Murashige and Skoog's medium with 2.23 μM naphthalene acetic acid. Maximum growth of cultured roots was 6.5 g fresh weight 40 ml^-1, as recorded on day 20. Even though normal roots were unable to accumulate capsaicin, they contained other phenylpropanoid intermediates and vanillylamine, as detected by HPLC analysis. Normal roots of Capsicum frutescens were treated with ferulic acid and protocatechuic aldehyde in order to study their biotransformation ability. Ferulic acid, which is the nearest precursor to vanillin, when fed at concentrations of 1 and 2 mM led to the accumulation of vanilla flavour metabolites, vanillin being the major one. In cultures treated with 1 and 2 mM ferulic acid, maximum vanillin accumulation of 12.3 and 16.4 μM was observed, on day 6 after precursor addition, respectively. Feeding of ferulic acid and β-cyclodextrin complex (2 mM each) enhanced the accumulation of biotransformed products. Moreover, vanillin accumulation was recorded as 24.7 μM on day 6 after precursor addition, which was 1.5 times higher than in cultures fed with ferulic acid (2 mM) alone. When ferulic acid was fed along with β-cyclodextrin (1 mM each) to cultures growing in a three-litre bubble column bioreactor, the maximum vanillin production of 10.7 μM was obtained; other vanilla flavour metabolites were also formed after 9 days of precursor addition. Root cultures could also biotransform protocatechuic aldehyde wherein a maximum vanillin production of 7.9 μM was recorded on day 6 after precursor addition. The bioconversion efficiency was observed to be 5–7% in case of ferulic acid fed cultures and 3.2% in case of protocatechuic aldehyde fed cultures suggesting the possible channelling of precursors to alternate biosynthetic pathways such as lignin.     

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.