Production Of Caffeic Acid Research Articles

Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3′-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3ʹ-hydroxylase (LaeC3′H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3′H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications.

Development of a co-culture system for green production of caffeic acid from sugarcane bagasse hydrolysate.

Caffeic acid (CA) is a phenolic acid compound widely used in pharmaceutical and food applications. However, the efficient synthesis of CA is usually limited by the resources of individual microbial platforms. Here, a cross-kingdom microbial consortium was developed to synthesize CA from sugarcane bagasse hydrolysate using Escherichia coli and Candida glycerinogenes as chassis. In the upstream E. coli module, shikimate accumulation was improved by intensifying the shikimate synthesis pathway and blocking shikimate metabolism to provide precursors for the downstream CA synthesis module. In the downstream C. glycerinogenes module, conversion of p-coumaric acid to CA was improved by increasing the supply of the cytoplasmic cofactor FAD(H2). Further, overexpression of ABC transporter-related genes promoted efflux of CA and enhanced strain resistance to CA, significantly increasing CA titer from 103.8 mg/L to 346.5 mg/L. Subsequently, optimization of the inoculation ratio of strains SA-Ec4 and CA-Cg27 in this cross-kingdom microbial consortium resulted in an increase in CA titer to 871.9 mg/L, which was 151.6% higher compared to the monoculture strain CA-Cg27. Ultimately, 2311.6 and 1943.2 mg/L of CA were obtained by optimization of the co-culture system in a 5 L bioreactor using mixed sugar and sugarcane bagasse hydrolysate, respectively, with 17.2-fold and 14.6-fold enhancement compared to the starting strain. The cross-kingdom microbial consortium developed in this study provides a reference for the production of other aromatic compounds from inexpensive raw materials.

Dynamic analysis Caffeic acid production driven by the key physicochemical factor and microbial community succession in Baijiu Daqu: A multi-microorganism fermentation of solid-state fermentation system

Daqu is a saccharifying fermenting agent in the solid-state fermentation process of Strong aromatic Baijiu, providing important raw materials, microorganisms and enzymes for the fermentation of Baijiu, however, a little information about organic acids production in Daqu until now. This work analyzed the correlation among microbial community succession, physicochemical factors and caffeic acid-producing metabolic pathways in key fermentation time-point of strong aromatic Baijiu Daqu, and the results showed that the main functional communities of caffeic acid metabolism were Staphylococcus, Klebsiella, Lactobacillus and Pichia, respectively. The correlation results showed that in the fermentation stage of the Daqu, caffeic acid was mainly influenced by miosture and reducing sugar. Co-occurrence network analysis showed that microbial interactions were stronger in days 0–8 than in days 10–27. The PICRUSt2 marker gene sequence was used to predict information on the function and abundance of enzymes related to different fermentation stages of Daqu, and combined with the related enzymes involve in caffeic acid metabolism, the results indicated that the microbial community in the pre-fermentation 0–8 days had the potential to produce caffeic acid. The results provide a theoretical basis for the analysis of the formation mechanism of phenolic acids in the multi-bacterial co-fermentation system of Daqu fermentation.

Caffeic acid production from glucose using metabolically engineered Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) is a precursor for high-valued compounds with anticancer, antiviral activities, and anti-inflammatory making it an important substance in the food additive, cosmetics, and pharmaceutical industries. Here, we developed an engineered Escherichia coli strain capable of directly producing high levels of caffeic acid from glucose. Tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) and p-coumaric acid 3-hydroxylase from Saccharothrix espanaensis (SeC3H) were expressed. Next, feedback-resistant chorismate mutase/prephenate dehydrogenase, was introduced to promote l-tyrosine synthesis. This engineered strain CA3 produced 1.58 g/L of caffeic acid from glucose without tyrosine supplemented to the medium. Furthermore, to reduce p-coumaric acid accumulation, 4-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa (PaHpaBC) was introduced. Finally, an engineered strain CA8 directly produced 6.17 g/L of caffeic acid from glucose using a jar fermenter. The E. coli developed in this study would be helpful as a chassis strain to produce value-added caffeic acid-derivatives.

Enhancing caffeic acid production in Escherichia coli by engineering the biosynthesis pathway and transporter

Caffeic acid is a phenylpropanoid which is widely used in medical industry. Microbial fermentation provides a green strategy for producing caffeic acid. To improve the capacity for caffeic acid production in Escherichia coli, the competing pathways for l-tyrosine synthesis were knocked out. The biosynthesis pathway of the cofactor FAD and the expression of previously reported polyphenol transporters were enhanced to promote the production of caffeic acid. Transcriptomics analysis was conducted to mine potential transporters that could further enhance the titer of caffeic acid in engineered E. coli. Transcriptomics data of E. coli under caffeic acid and ferulic acid stress showed that 19 transporters were upregulated. Among them, overexpression of ycjP, which was previously identified as a sugar ABC transporter permease, improved the caffeic acid titer to 775.7 mg/L. The caffeic acid titer was further improved to 7922.0 mg/L in a 5-L fermenter, the highest titer achieved by microorganisms.

High-density fermentation of Escherichia coli to express 4-hydroxyphenylacetate 3-hydroxylase and efficient biosynthesis of caffeic acid

The 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H), originated from Escherichia coli, converts p-coumaric acid to caffeic acid. In order to improve the efficiency of caffeic acid biosynthesis, we engineered E. coli for overexpression of 4HPA3H. The high-density fermentation of the engineered E. coli was conducted in a 5 L bioreactor. Subsequently, the conditions for whole-cell biocatalysis were optimized. The dry cell weight of the 4HPA3H-expressed strain reached 34.80 g/L. After incubated in the bioreactor for 6 h, 18.74 g/L (0.85 g/(L·OD600)) of caffeic acid was obtained, with a conversion rate of 78.81% achieved. To the best of our knowledge, the titer of caffeic acid is the highest reported to date. The high-density fermentation of E. coli for overexpression of 4HPA3H and the efficient biosynthesis of caffeic acid may facilitate future large-scale production of caffeic acid.

Influence of biotic and abiotic elicitors on artemisinin, quercetin, caffeic acid and essential oil production in Artemisia dracunculus L.

AbstractArtemisia dracunculus L. (tarragon) is valuable for its medicinal uses such as antimalarial, antibacterial and anticancer. Biotechnological methods need to be developed for the production of phytochemical compounds in plants with high and stable quality. Elicitation is one of the best tools for increasing secondary metabolites in various in vitro cultures. The current study determines the application of different concentrations of chitosan (25, 50, 100 and 150 mg/L) and UV‐C (5/10, 5/15, 10/10 and 10/15 min/cm) as biotic and abiotic elicitors in tarragon leaf callus cultures. The main aim was to increase the accumulation of artemisinin, quercetin, caffeic acid and essential oil content. For this purpose, callus formation rates and morphological features were also investigated. The results show that highest callus formation, weight and diameter were observed in UV‐C treated cultures. HPLC analyses revealed significant high accumulation of artemisinin (1.9 μg/g), quercetin (4.2 μg/g) and caffeic acid (2.9 μg/g) contents in 5/10 min/cm UV‐C treated samples. GC–MS analyses of callus cultures indicate high production of total amount of monoterpenes in all chitosan applications, best in 25 mg/L (90.75%). The major compound was β‐phellandrene in chemical composition of tarragon (19.61%) and increased the most in 10/15 min/cm UV‐C application (43.39%). The results show that the physical elicitor UV‐C can be effectively used in tarragon callus culture for enhancing pharmacologically active compounds in industrial production.

De novo biosynthesis of p-coumaric acid and caffeic acid from carboxymethyl-cellulose by microbial co-culture strategy

BackgroundAromatic compounds, such as p-coumaric acid (p-CA) and caffeic acid, are secondary metabolites of various plants, and are widely used in diet and industry for their biological activities. In addition to expensive and unsustainable methods of plant extraction and chemical synthesis, the strategy for heterologous synthesis of aromatic compounds in microorganisms has received much attention. As the most abundant renewable resource in the world, lignocellulose is an economical and environmentally friendly alternative to edible, high-cost carbon sources such as glucose.ResultsIn the present study, carboxymethyl-cellulose (CMC) was utilized as the sole carbon source, and a metabolically engineered Saccharomyces cerevisiae strain SK10-3 was co-cultured with other recombinant S. cerevisiae strains to achieve the bioconversion of value-added products from CMC. By optimizing the inoculation ratio, interval time, and carbon source content, the final titer of p-CA in 30 g/L CMC medium was increased to 71.71 mg/L, which was 155.9-fold higher than that achieved in mono-culture. The de novo biosynthesis of caffeic acid in the CMC medium was also achieved through a three-strain co-cultivation. Caffeic acid production was up to 16.91 mg/L after optimizing the inoculation ratio of these strains.ConclusionDe novo biosynthesis of p-CA and caffeic acid from lignocellulose through a co-cultivation strategy was achieved for the first time. This study provides favorable support for the biosynthesis of more high value-added products from economical substrates. In addition, the multi-strain co-culture strategy can effectively improve the final titer of the target products, which has high application potential in the field of industrial production.

Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast.

Advances in synthetic biology enable microbial hosts to synthesize valuable natural products in an efficient, cost-competitive and safe manner. However, current engineering endeavors focus mainly on enzyme engineering and pathway optimization, leaving the role of cofactors in microbial production of natural products and cofactor engineering largely ignored. Here we systematically engineered the supply and recycling of three cofactors (FADH2, S-adenosyl-L-methion and NADPH) in the yeast Saccharomyces cerevisiae, for high-level production of the phenolic acids caffeic acid and ferulic acid, the precursors of many pharmaceutical molecules. Tailored engineering strategies were developed for rewiring biosynthesis, compartmentalization and recycling of the cofactors, which enabled the highest production of caffeic acid (5.5 ± 0.2 g l-1) and ferulic acid (3.8 ± 0.3 g l-1) in microbial cell factories. These results demonstrate that cofactors play an essential role in driving natural product biosynthesis and the engineering strategies described here can be easily adopted for regulating the metabolism of other cofactors.

Developing Multi-Copy Chromosomal Integration Strategies for Heterologous Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae.

Caffeic acid, a plant-sourced phenolic compound, has a variety of biological activities, such as antioxidant and antimicrobial properties. The caffeic acid biosynthetic pathway was initially constructed in S. cerevisiae, using codon-optimized TAL (coTAL, encoding tyrosine ammonia lyase) from Rhodobacter capsulatus, coC3H (encoding p-coumaric acid 3-hydroxylase) and coCPR1 (encoding cytochrome P450 reductase 1) from Arabidopsis thaliana in 2 μ multi-copy plasmids to produce caffeic acid from glucose. Then, integrated expression of coTAL via delta integration with the POT1 gene (encoding triose phosphate isomerase) as selection marker and episomal expression of coC3H, coCPR1 using the episomal plasmid pLC-c3 were combined, and caffeic acid production was proved to be improved. Next, the delta and rDNA multi-copy integration methods were applied to integrate the genes coC3H and coCPR1 into the chromosome of high p-coumaric acid yielding strain QT3-20. The strain D9 constructed via delta integration outperformed the other strains, leading to 50-fold increased caffeic acid production in optimized rich media compared with the initial construct. The intercomparison between three alternative multi-copy strategies for de novo synthesis of caffeic acid in S. cerevisiae suggested that delta-integration was effective in improving caffeic acid productivity, providing a promising strategy for the production of valuable bio-based chemicals in recombinant S. cerevisiae.

Production of Caffeic Acid with Co-fermentation of Xylose and Glucose by Multi-modular Engineering in Candida glycerinogenes.

Caffeic acid (CA), a natural phenolic compound, has important medicinal value and market potential. In this study, we report a metabolic engineering strategy for the biosynthesis of CA in Candida glycerinogenes using xylose and glucose. The availability of precursors was increased by optimization of the shikimate (SA) pathway and the aromatic amino acid pathway. Subsequently, the carbon flux into the SA pathway was maximized by introducing a xylose metabolic pathway and optimizing the xylose assimilation pathway. Eventually, a high yielding strain CG19 was obtained, which reached a yield of 4.61 mg/g CA from mixed sugar, which was 1.2-fold higher than that of glucose. The CA titer in the 5 L bioreactor reached 431.45 mg/L with a yield of 8.63 mg/g of mixed sugar. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of CA. This is the first report to produce CA in C. glycerinogenes with xylose and glucose as carbon sources, which developed a promising strategy for the efficient production of high-value aromatic compounds.

Synergistic effects of salicylic acid and light stress on bioactive metabolites in basil callus cultures

Thai basil is a well-known medicinal plant that is rich in bioactive antioxidant compounds having a variety of health benefits, including cancer, diabetes, and cardiovascular disease prevention. Plant cell and tissue culture technologies can be systematically developed as a valuable, low-cost biomass supply for the production of high-value phytochemicals. The aim of this study is to create an effective protocol for producing “Thai basil” leaf derived callus cultures with a high yield of biomass and antioxidants as an alternative to growing plants using salicylic acid and various light regimes as elicitors. Salicylic acid (SA) and light significantly affects many metabolic processes coupled with biosynthesis of secondary metabolites in plants. Callus was exploited with various SA concentrations under 3 different light regimes. Indeed, the results of our research disclose that continuous light with moderate SA concentration (10 μM) significantly increased total phenolic content (18.7 ± 1.13 mg/g DW), flavonoid content (7.2 ± 0.21 mg/g DW), eugenol (0.56 ± 0.015 mg/g DW), rosmarinic acid (54.35 ± 1.85 mg/g DW), chicoric acid (64.46 ± 6.71 mg/g DW) cyanidin (0.42 ± 0.021 mg/g DW) and peonidin (0.32 ± 0.002 mg/g DW) production followed by photoperiod and dark. SA treatment (25 μM) under photoperiod showed maximum biomass accumulation (FW: 226.6 ± 3.91 g/L and DW: 16.44 ± 0.59 g/L) and caffeic acid production (0.54 ± 0.025 mg/g DW). Moreover in vitro antioxidant potential (DPPH and ABTS) and cellular antioxidant capacity was found higher in continuous light treated callus along with 10 μM SA treatment. Callus extracts also showed biocompatibility with human erythrocytes that indicates their nontoxic nature and moderate potential against alpha-amylase inhibition which will help in regulating glucose level. Compared to commercial leaves, callus extracts showed higher production of chicoric acid and rosmarinic acid associated with higher antioxidant capacity. In addition, this biological system also has a large capacity for continuous biomass production, thus demonstrating its high potential for possible nutraceutical applications.

A chlorogenic acid esterase from a metagenomic library with unique substrate specificity and its application in caffeic and ferulic acid production from agricultural byproducts

Soil microbes are an abundant source of enzymes with unique properties that may be useful for industrial applications. As most wild-type strains show low chlorogenic acid esterase expression and activity, and most microbes cannot be cultured in the laboratory, a metagenomic approach provides methods of identifying new enzymes. In this study, a gene encoding a chlorogenic acid esterase, named Tan410, was isolated from a soil metagenomic library and overexpressed in Escherichia coli BL21 (DE3). The recombinant enzyme, with a predicted molecular weight of 54.88 kDa, was purified to homogeneity. The K m and V max values for Tan410 were 1.26 mM and 0.33 mM min–1, respectively, with chlorogenic acid as the substrate. Its optimum temperature and pH for reaction were 30 °C and 7.5, respectively. The enzyme exhibited moderate thermostability and broad pH stability (3.0–10.0). Tan410 was also able to hydrolyse ethyl ferulate, methyl caffeate, propyl gallate, ethyl gallate, methyl vanillate, methyl benzoate, ethyl benzoate, methyl 2,5-dihydroxybenzoate, and methyl 3,5-dihydroxybenzoate, and it released caffeic and ferulic acids from agricultural byproducts (destarched wheat bran and coffee pulp). Tan140 has potential for industrial application in biomass valorization.

Metabolic engineering of Saccharomyces cerevisiae for enhanced production of caffeic acid.

As a natural phenolic acid product of plant source, caffeic acid displays diverse biological activities and acts as an important precursor for the synthesis of other valuable compounds. Limitations in chemical synthesis or plant extraction of caffeic acid trigger interest in its microbial biosynthesis. Recently, Saccharomyces cerevisiae has been reported for the biosynthesis of caffeic acid via episomal plasmid-mediated expression of pathway genes. However, the production was far from satisfactory and even relied on the addition of precursor. In this study, we first established a controllable and stable caffeic acid pathway by employing a modified GAL regulatory system to control the genome-integrated pathway genes in S. cerevisiae and realized biosynthesis of 222.7 mg/L caffeic acid. Combinatorial engineering strategies including eliminating the tyrosine-induced feedback inhibition, deleting genes involved in competing pathways, and overexpressing rate-limiting enzymes led to about 2.6-fold improvement in the caffeic acid production, reaching up to 569.0 mg/L in shake-flask cultures. To our knowledge, this is the highest ever reported titer of caffeic acid synthesized by engineered yeast. This work showed the prospect for microbial biosynthesis of caffeic acid and laid the foundation for constructing biosynthetic pathways of its derived metabolites. KEY POINTS: Genomic integration of ORgTAL, OHpaB, and HpaC for caffeic acid production in yeast. Feedback inhibition elimination and Aro10 deletion improved caffeic acid production. The highest ever reported titer (569.0 mg/L) of caffeic acid synthesized by yeast.

Transcriptomic and metabolomic profiling reveals the effect of LED light quality on morphological traits, and phenylpropanoid-derived compounds accumulation in Sarcandra glabra seedlings

BackgroundSarcandra glabra is an evergreen and traditional Chinese herb with anti-oxidant, anti-bacterial, anti-inflammatory, and anti-tumor effects. Light is one of the most influential factor affecting the growth and quality of herbs. In recent times, the introduction of Light Emission Diode (LED) technology has been widely used for plants in greenhouse. However, the impact of such lights on plant growth and the regulatory mechanism of phenylpropanoid-derived compounds in S. glabra remain unclear.ResultsThe red LED light (RL) substantially increased the plant height and decreased the stem diameter and leaf area relative to the white LED light (WL), while the blue LED light (BL) significantly reduced the height and leaf area of S. glabra. According to transcriptomic profiling, 861, 378, 47, 10,033, 7917, and 6379 differentially expressed genes (DEGs) were identified among the groups of leaf tissue under BL (BY) vs. leaf tissue under RL (RY), BY vs. leaf tissue under WL (WY), RY vs. WY, root tissue under WL (WG) vs. WY, stem tissue under WL (WJ) vs. WG, and WJ vs. WY, respectively. We identified 46 genes encoding for almost all known enzymes involved in phenylpropanoid biosynthesis, e.g., phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), and flavonol synthase (FLS). We found 53 genes encoding R2R3-MYB proteins and bHLH proteins, respectively, where several were related to flavonoids biosynthesis. A total of 454 metabolites were identified based on metabolomic profiling, of which 44, 87, and 296 compounds were differentially produced in WY vs. RY, WY vs. BY, and WY vs. WG. In BY there was a substantial reduction in the production of esculetin, caffeic acid, isofraxidin, and fraxidin, while the yields of quercitrin and kaempferol were significantly up-regulated. In RY, the contents of cryptochlorogenic acid, cinnamic acid, and kaempferol decreased significantly. Besides, in WG, the production of metabolites (e.g. chlorogenic acid, cryptochlorogenic acid, and scopolin) declined, while their yields increased significantly (e.g. esculetin, fraxetin, isofraxidin, and fraxidin).ConclusionThese results provide further insight into the regulatory mechanism of accumulation patterns of phenylpropanoid-derived compounds in S. glabra under various light conditions, allowing optimum breeding conditions to be developed for this plant.

Increased phenolic acid and tanshinone production and transcriptional responses of biosynthetic genes in hairy root cultures of Salvia przewalskii Maxim. treated with methyl jasmonate and salicylic acid.

The purpose of this study is to reveal the impact of the plant hormone salicylic acid (SA) and methyl jasmonate (MeJA) on the growth, effective components accumulation, and related gene expression of the hairy root of Salvia przewalskii Maxim. Various concentrations of SA (0, 25, 50, 100, 200μM) or MeJA (0, 50, 100, 200, 400, 600μM) were added to the culture medium of Salvia przewalskii Maxim. Low concentrations of SA promoted the growth of hairy root, while a high concentration inhibited it. 0 to 400μM MeJA promoted the growth of hairy root, but 600μM MeJA starts to inhibit its growth. 50μM SA and 400μM MeJA significantly enhanced the production of caffeic acid, rosmarinic acid, salvianolic acid B, cryptotanshinone, and tanshinone IIA. In general, 50μM SA can be used to accumulate of tanshinone in hairy roots of S. przewalskii with 6days. 400μM MeJA can be used to accumulate of phenolic acids in hairy roots of S. przewalskii with 3days. The selected genes in the tanshinone and phenolic acid biosynthetic pathway were upregulated with elicitation. To obtain a higher yield and content of secondary metabolites, it is advisable to use 50μM SA or 400μM MeJA as the optimal doses to cultivate the hairy root of S. przewalskii. This study provides, for the first time, an efficient tanshinone and phenolic acid production method for S. przewalskii.

Interactive Effects of Wide-Spectrum Monochromatic Lights on Phytochemical Production, Antioxidant and Biological Activities of Solanum xanthocarpum Callus Cultures.

Solanum xanthocarpum is considered an important traditional medicinal herb because of its unique antioxidant, and anti-diabetic, anti-aging, and anti-inflammatory potential. Because of the over exploitation linked to its medicinal properties as well as destruction of its natural habitat, S. xanthocarpum is now becoming endangered and its supply is limited. Plant in vitro culture and elicitation are attractive alternative strategies to produce biomass and stimulate biosynthesis of medicinally important phytochemicals. Here, we investigated the potential influence of seven different monochromatic light treatments on biomass and secondary metabolites accumulation in callus culture of S. xanthocarpum as well as associated biological activities of the corresponding extracts. Among different light treatments, highest biomass accumulation was observed in white light-treated callus culture. Optimum accumulation of total flavonoid contents (TFC) and total phenolic contents (TPC) were observed in callus culture kept under continuous white and blue light respectively than control. Quantification of phytochemicals through HPLC revealed that optimum production of caffeic acid (0.57 ± 0.06 mg/g DW), methyl-caffeate (17.19 mg/g ± 1.79 DW), scopoletin (2.28 ± 0.13 mg/g DW), and esculetin (0.68 ± 0.07 mg/g DW) was observed under blue light callus cultures. Compared to the classic photoperiod condition, caffeic acid, methyl-caffeate, scopoletin, and esculetin were accumulated 1.7, 2.5, 1.1, and 1.09-folds higher, respectively. Moreover, high in vitro cell free antioxidant, anti-diabetic, anti-aging, and anti-inflammatory activities were closely associated with the production of these secondary metabolites. These results clearly showed the interest to apply multispectral light as elicitor of in vitro callus cultures S. xanthocarpum to promote the production of important phytochemicals, and allow us to propose this system as an alternative for the collection of this endangered species from the wild.

Efficient plant regeneration and callus induction from nodal and hypocotyl explants of goji berry (Lycium barbarum L.) and comparison of phenolic profiles in calli formed under different combinations of plant growth regulators

Goji berry (Lycium barbarum L.) is a fruit that has many beneficial effects (such as antidiabetic, antioxidant, anticancer, antidepressant, and immunomodulatory) on human health because of several dietary constituents such as phenolics, vitamins, carotenoids, and polysaccharides. In order to develop in vitro culture protocols for callus induction and plant regeneration from different explants of Goji plants and to compare the phenolic composition in calli of different origin, various combinations of plant growth regulators (PGRs) were applied. Various types of explants (leaf, petiole, root, hypocotyl, and node) were cultured on MS medium containing 28 different concentrations and combinations of PGRs [thidiazuron (TDZ) and naphthalene acetic acid (NAA), TDZ and indole-3-acetic acid (IAA), benzyl adenine (BA) and NAA, alone TDZ, and alone BA]. The highest mean number of shoots (23.33 ± 1.86) and percentage of shoot formation (100%) were obtained from nodal explants on medium containing 0.5 mg/l BA alone. The highest mean callus diameter was obtained from hypocotyl explants on medium containing 0.25 mg/l TDZ and 0.1 mg/l IAA (21.40 ± 0.71 mm). The amounts of selected phenolic substances were significantly different in the callus obtained from different PGRs or combinations, individually. The combination of BA/NAA significantly increased the production and accumulation of chlorogenic acid and caffeic acid. The TDZ/IAA combination, TDZ alone, and TDZ/NAA combination significantly increased vanillic acid and rutin, gallic acid, and quercetin synthesis, respectively. These outcomes indicate that different PGRs lead to the production of different kinds of secondary metabolites and affect/accelerate accumulation in the callus of goji berry. The culture protocol described in this paper could be employed for the development of novel methods for the commercial production of goji secondary metabolites.

Kinetic identification of phenolic compounds and potential production of caffeic acid by Ganoderma lipsiense in solid-state fermentation.

Basidiomycetes fungi have been utilized for the production of several compounds with bioactive properties, such as phenolic compounds. The present work quantified and identified the phenolic compounds produced in a kinetic study (63days) and evaluated the antimicrobial activity from the extract obtained by Ganoderma lipsiense cultivation in solid-state fermentation using red rice. Phenolic compounds were identified by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and caffeic acid content was measured by high-performance liquid chromatography with diode-array detection (HPLC-DAD). Caffeic and syringic acids were produced by G. lipsiense. In the control medium (red rice), the following compounds were identified: p-coumaric acid, salicylic acid, ferulic acid and vanillin. High concentrations of caffeic acid (0.977µgg-1) were measured in 49days. Antimicrobial activity was investigated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus using a minimum inhibitory concentration (MIC) technique. Ganoderma lipsiense extract was only effective against P. aeruginosa. These data have proved to be satisfactory in the study of biosynthesis of caffeic acid and antibacterial compounds by G. lipsiense in solid-state fermentation with red rice.

Engineering the Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae with Heterologous Enzyme Combinations

Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg·L−1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.