Phenolic amides (avenanthramides) in oats – an update review

ABSTRACT

Oats (Avena sativa L.) are one of the worldwide cereal crops. Avenanthramides (AVNs), the unique plant alkaloids of secondary metabolites found in oats, are nutritionally important for humans and animals. Numerous bioactivities of AVNs have been investigated and demonstrated in vivo and in vitro. Despite all these, researchers from all over the world are taking efforts to learn more knowledge about AVNs. In this work, we highlighted the recent updated findings that have increased our understanding of AVNs bioactivity, distribution, and especially the AVNs biosynthesis. Since the limits content of AVNs in oats strictly hinders the demand, understanding the mechanisms underlying AVN biosynthesis is important not only for developing a renewable, sustainable, and environmentally friendly source in both plants and microorganisms but also for designing effective strategies for enhancing their production via induction and metabolic engineering. Future directions for improving AVN production in native producers and heterologous systems for food and feed use are also discussed. This summary will provide a broad view of these specific natural products from oats.

Highlights

• Avenanthramides are unique nutritional alkaloids in oats

• AVN bioactivity, distribution, and the potential AVNs biosynthesis are discussed

• AVNs can be produced via induction and metabolic engineering

Keywords:

• Avenanthramide
• Avena sativa L.
• biosynthesis
• bioactivity
• metabolic
• engineering
• hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyltransferase

1. Introduction

Plant secondary metabolites are non-essential, small-molecule organic compounds produced via secondary metabolism for plant growth and development [1,2]. They are vital in adaptation to environmental stress, competition, and co-evolution among plants, insects, herbivores, and pathogenic microorganisms by playing physiological roles in signal transduction, nutrition, stress resistance, and host defense [3–5]. Many plant metabolites including phenolic acids, alkylphenols, flavonoids, lignans aminophenolics and avenanthramides have various pharmacological effects on human health and well-being [3–8].

Oats (Avena sativa L.) are cereal crops with the same origin as medicines and food. They rank sixth in terms of cereal production worldwide, followed by wheat, maize, rice, barley, and sorghum. They have a long history of planting worldwide and are found in mountainous areas, plateaus, and northern alpine regions, adapting to lower growth temperatures and exhibiting strong drought resistance, salt-alkali resistance, and a short growth period [9,10]. Depending on whether the seeds are shelled, there are two types of oats: hulled and naked [11]. Oats are mainly used as a traditional food crop and feed and have extremely high nutritional content. They are rich in dietary fiber, crude protein, trace elements, amino acids required for protein synthesis, high-quality oils, and many antioxidant substances compared with other cereal crops [12–17]. Among oats antioxidant substances, avenanthramides (AVNs), which is the unique bioactive compounds in oat, present greater antioxidant activities than other antioxidants and process anti-proliferative, vasodilative, cytoprotective, antioxidant, anti-hyperglycemic and anti-inflammatory properties. Therefore, these compounds are widely used in foods preparation, cosmetic products and supplements [10,13,18–26]

In recent years, some reviews about their biosynthesis, distribution, activity and diverse functions have been reported [13,27–30]. This indicates that increasing attention and demand are required for the knowledge of AVNs in oats. With the respects of those previous reviews and studies on AVNs, we here proposed the latest summary. In this review, structural and functional diversity and distribution of these unique oat phytochemicals are introduced, and the update information of their biosynthesis mechanism, methods for enhancing AVNs content and potential strategies for effective metabolic engineering of these AVNs in oats or heterologous systems for food and feed applications are discussed in the following section.

2. Avenanthramides

Oats are rich in antioxidants, including phenolic acids, flavonoids, vitamin E, and avenanthramides [31]. Among these nutritional compounds, oat alkaloids, also known as oat polyphenols, are secondary metabolites unique to oats. These alkaloids levels are higher than the contents of phenolic acids and are considered to be the main antioxidant compounds of oats [13,32–34]. Oat alkaloids were defined as phytoalexins, which function on resisting crown rust (Puccinia coronata) infection. Later, more evidences showed that these compounds play the key role on defending against pathogens, including insects, fungi, and other environmental stresses [29].

Compared to the other cereals, oats contain a unique group of phenolic alkaloids known as avenanthramides (AVNs). Structural analysis showed that AVNs are conjugates of one of three phenylpropanoids (p-coumaric, ferulic, or caffeic acids) and anthranilic acid (or a hydroxylated and/or methoxylated derivative of anthranilic acid) (Figure 1). AVNs have a high UV-absorbing ability [35,36]. More than 40 forms of AVN have recently been identified and named, according to Collins and Dimberg. Collins named avenalumin-like compounds AVNs and assigned an alphabetical name to each AVN congener [35]. Later, Dimberg et al. modified the systematic nomenclature system by using an upper-case letter to represent anthranilate derivatives (e.g. A = anthranilate, B = 5-hydroxy anthranilate) and a lower-case letter to represent phenylpropionates (e.g. c = caffeic acid, p = p-coumaric, s = sinapic acid, f = ferulic acid, or a = cinnamic acid) [37]. For AVNs with longer phenylpropionates, additional subscripted letters have been added to clarify their structures.

Figure 1. Chemical structure of avenanthramides from previous studies [35–37].

3. Distribution of AVNs in oat

AVNs are secondary metabolites, and the types, locations, and accumulation levels vary in different plant tissues, species, growth stages, growth conditions, and locations. Generally, as shown in Figure 2a, oat bran and flakes contain higher amounts of AVNs (1.3–12.5 mg/100 g) relative to the other tissues, therefore, AVNs are always present in all milling fractions and commercial oat products. For example, the crude milling oat products, including oat grains, oat flour, oat bran, oat cereal, and oat bread contain around 37–45, 33–70, 33–94, 25–78, and 5–6 µg/g AVNs, respectively (Figure 2b) [38]. Refined oat products like extruded breakfast cereal, oat bread, and oat pasta contain fewer AVNs, ranging from 2 to 30 µg/g compared to crude milling oat products [39]. Therefore, consuming whole grain oats is better than refined grains for absorbing more AVNs.

Figure 2. Distribution of avenanthramides (AVNs) in oat. (a) Distribution of AVNs in different oat tissues [38,39]. (b) Distribution of AVNs in different oat cultivars [37,40]. (c) Distribution of AVNs in different growth stages [34,41–48]. (d) Potential factors on changing levels of AVNs [41,49].

In addition, different oat organs contain different AVN levels. Peterson and Dimberg measured AVN levels in oat spikelets and leaves at various growth stages [37]. As the results, AVNs generally increased throughout maturation in which the AVN concentrations in spikelets were generally higher than those in leaves. Later, Canadian researchers analyzed AVN levels in oat tissues and found that AVNs were produced in both vegetative tissues and grains. Gene expression analysis of target gene indicated that leaf is the predominant location for AVNs biosynthesis. However, higher AVNs amounts were found in the upper and lower stems, roots, panicle stems, and glumes, while lemma, palea, and filling grains contained substantially lower amounts of AVNs [40]. Moreover, AVNs could be detected in phloem sap. These findings indicated that the AVNs were first synthesized in leaf and then transported to glumes and other tissues.

The AVN content also varies among different cultivars. Multari et al. investigated the concentrations of AVNs in eight Finnish husked oat cultivars. The total AVN levels ranged from 26.7 ± 1.44 to 185 ± 12.5 mg/kg among these cultivars. In detail, the cultivar ‘Avetron’ presented the lowest total AVN content, while ‘Viviana’ showed the highest total AVN content, 6.9-fold higher than ‘Avetron’ [50]. Dvořáček et al. compared AVN contents from five oat cultivars, concluding that the total AVNs fluctuated from 25 to 407 mg/kg dry weight, and the level of relative variability of individual AVNs varied from 72 to 114% [41]. Other summary of the AVNs content in different cultivars were listed in Table 1 [34,42–48]. The accumulation of AVN types and concentrations varies at different growth stages (Figure 2c). de Bruijn analyzed AVNs from oat seeds to seedlings and found that AVNs content is increased by germination, resulting in approximately 25 times larger quantities in oat seedlings [49]. In seedling extracts, AVN-A, AVN-B, and AVN-C represent less than 20% of the total AVN content, indicating that more AVNs were produced during germination. Moreover, grain AVNs production tends to be constitutive, but AVNs concentrations are highly variable and strongly influenced by environmental conditions and stress, including season, locality, and cropping system (Figure 2d). Higher levels of precipitation, especially in May and June, significantly increase the total AVNs in hulled and naked oats. The above-average temperatures in all three monitored years and higher precipitation amounts could have affected plant development and the beginning of grain formation, including AVNs synthesis [41]. Therefore, the optimal oat materials for AVNs extraction can be selected through cultivars, seasons and the processing methods for increase of the production. Especially, germination of oat seed could be an optimal treatment for enhancing AVNs in a short period.

Table 1. Levels of AVNs in different cultivars.

Cultivar	Avenanthramide A	Avenanthramide B	Avenanthramide C	Reference
Akseli	6.9	4.7	5.2
Avetron	6.0	4.8	3.5
Ivory	8.4	12.3	7.2
Marika	7.1	8.8	5.9	[50]
Peppi	20.9	18.5	19.0
Riina	6.8	4.4	3.5
Rocky	10.9	16.9	10.2
Viviana	29.6	21.9	39.2
Kertag	20.2	51.3	12.6
Korok	14.0	33.3	11.1
Patrik	26.3	54.5	21.6	[41]
Raven	17.8	48.7	16.6
Seldon	24.6	48.2	20.0
Swedish cultivars (Kapp, Mustang, Svea)	25.0–47.0	21.0–43.0	28.0–62.0	[42]
Three U.S. cultivars, groats	12.0–22.5	22.2–41.2	37.1–65.7	[34]
Sang	18.1	8.8	12.0	[43]
Bullion	140.0	124.5	79.0
Bikini	36.4	36.1	30.6
Ripon	36.1	21.8	24.7
Neon	31.1	20.5	30.9
Three Swedish cultivars	14.7–50.8	11.8–34.5	15.4–51.0	[44]
Four Turkish genotypes	3.5–16.8	3.4–11.4	6.5–16.8	[45]
Japanese (Shokan 1)	22.9	26.6	44.1	[46]
33 different U.S. genotypes	1.2–5.6	1.0–5.0	1.4–5.4	[47]
Dancer	19.8	15.1	29.5	[48]
Leggett	18.7	14.2	31.8
Morgan	17.8	15.4	29.2
Nicolas	16.3	12.3	35.9
Orrin	21.4	18.5	26.6
Souris	18.3	14.4	24.8

4. Functions of AVN as a plant bioactive compound

Many studies revealed that AVNs have specific functions in human health and well-being. The bioactive compounds present have nutraceutical properties such as antioxidant, bioavailability, anti-inflammatory, antiproliferative, vasodilation, and anti-itch effects (Figure 3, Table 2).

Figure 3. Summary of health benefit effects of avenanthramides [15,24,49,51–65].

Table 2. Representative bioactive properties of AVNs for health.

biological potential	Model of action	References
	Inhibition of β-carotene bleaching	[22,51]
	Reaction with the free radical DPPH
Antioxidant	Enhancement of superoxide dismutase and glutathione peroxidase activity	[51]
	Increase of the plasma concentration of reduced glutathione	[52]
	Inhibition of galactose-induced oxidative stress and lipoxygenase activity	[53,54]
	Inhibition of the IL-1β-stimulated endothelial cell secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8 and MCP-1)	[49]
Anti-inflammatory	Suppression of proinflammatory cytokine production	[15]
	Decrease of neutrophil respiratory burst, NFκB activation, plasma IL-6 concentration, and erythrocyte glutathione peroxidase activity and increased reduced glutathione levels	[57]
	Inhibition of passive cutaneous anaphylactic reactions	[27]
Antiproliferation	Inhibition of cancer cells development	[57]
Anti-irritant	Protection of normal human skin fibroblasts	[60]
	Inhibition of NF-κB and activation of Nrf2/HO-1	[61,62]
Others	Reduction of blood pressure and the risk of cardiovascular disease	[63–65]
	Protection pancreatic βcells, the insulinproducing cells, from cytokines	[12]

4.1 Antioxidant activity

Most AVNs from oat exhibit potent antioxidant properties. And the antioxidant activity of AVNs is significantly greater than that of other phenolic antioxidants, such as vanillin and caffeic acid. Two in vitro systems, including the inhibition of β-carotene bleaching, reaction with the free radical 2,2-diphenyl-1-picrylhy-drazyl (DPPH), and ferric reducing antioxidant potential assay had been applied to test the antioxidant abilities of three major AVNs, AVN-A, AVN-B, and AVN-C [21,51]. The three AVNs displayed antioxidant activity in both systems, with AVN-C having the greatest activity. AVN-C was nearly as functional as the standard synthetic antioxidant, butylated hydroxytoluene in the β-carotene system. In the DPPH system, AVN-C and AVN-B were more active than the standard antioxidant 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox®). Additionally, AVN-C exhibited the highest antioxidant activity in the ferric reducing antioxidant potential assay.

The antioxidant activity of AVN-enriched oat extracts was also investigated in laboratory animals. Supplementing the diet of rats with 100 mg/kg AVN-enriched oat extracts (providing approximately 20 mg/kg AVNs) increases superoxide dismutase activity in the skeletal muscle, liver, and kidneys and enhances glutathione peroxidase activity in the heart and skeletal muscles [51]. The bioavailability and antioxidant actions of the major AVNs, including AVN-A, AVN-B, and AVN-C, have also been investigated in humans. At doses of 0.5 and 1.0 g of an AVN-enriched mixture, the AVN content reached the maximum peak in plasma at 1.5 and 2.3 h, respectively [52]. AVN-A and AVN-B bio-availabilities were 18- and 5-fold higher in humans than in hamsters, respectively. Interestingly, the consumption of AVN-enriched oat extracts significantly increased the plasma concentration of reduced glutathione, the master antioxidant in the body. Specifically, after consuming 0.1 g of AVN containing mixture, plasma glutathione levels increased by 21% from the baseline at 15 min, without apparent adverse side effects [52].

Moreover, AVN-rich oat extracts possess effective antioxidant activity against D-galactose-induced oxidative stress and inhibit lipoxygenase activity [53]. Twelve AVNs (0.6 mM) were used as the LOX inhibitors. The corresponding free cinnamic acids, the AVN analog Tranilast®, and the LOX inhibitor trans-resveratrol were included for comparison. AVN-A comprising caffeic or sinapic acid exhibited significant lipoxygenase inhibition (60–90%) (p < 0.05), whereas low or no inhibition was observed with AVN containing p-coumaric or ferulic acid. No difference in inhibition was observed when AVN was compared with their corresponding free cinnamic acid, implying that the anthranilic acid part of the AVN molecule does not affect inhibition. Trans-resveratrol showed inhibition, whereas no inhibition was seen for Tranilast® at the concentrations used in this study, suggesting that AVN containing caffeic or sinapic acid partially exert antioxidant effect via lipoxygenase inhibition [54]. Collectively, these results demonstrated that AVNs from oat possess strong antioxidant activity in animal cells.

4.2 Anti-inflammatory activity

In addition, in vivo and in vitro experiments have shown that AVNs have anti-inflammatory, anti-malignant cell proliferation, antipruritic, anti-coronary heart disease, anti-rectal cancer, and other effects [31,49,55,56] A study showed that application of oats extracts inhibit the IL-1β-stimulated endothelial cell secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8 and MCP-1), the expression of adhesion molecules (ICAM-1, VCAM-1, and E-selectin), and the adhesion of monocytes to the endothelial cell monolayer [49]. In a randomized, double-blind trial, different AVN levels (either 9.2 mg or 0.4 mg) exhibited different anti-inflammatory statuses in women after an 8-week dietary regimen [15]. Blood sample results showed that AVN supplementation (9.2 mg) significantly decreased the systemic inflammatory response, as assessed via measuring the neutrophil respiratory burst at 24 h and C-reactive protein level at 48 h. AVN supplementation also suppressed proinflammatory cytokine production, assessed by plasma interleukin (IL)-1β concentration and mononuclear cell nuclear factor-kappa B (NF-κB) binding. In a similar study, the same authors evaluated the effect of AVN supplementation on the systemic inflammatory response in younger women (18–30 years old) after exercise [57]. The results showed significantly decreased neutrophil respiratory burst, NFκB activation, plasma IL-6 concentration, and erythrocyte glutathione peroxidase activity and increased reduced glutathione levels after AVN supply. Thus, long-term AVN supplementation appears to be a useful dietary strategy to reduce inflammation after physical exercise in younger and older women. AVN-C (1–100 nM) suppresses phosphorylation of phosphoinositide 3-kinase and phospholipase Cγ1 and decreases intracellular calcium levels by inhibiting the immunoglobulin E (IgE)-stimulated mast cell degranulation. Oral administration of AVN-C attenuates active systemic anaphylactic reactions in a dose-dependent manner, as evidenced by the inhibition of hypothermia and reduction of elevated serum histamine, IgE, and interleukin-4 levels. AVN-C also inhibits passive cutaneous anaphylactic reactions, such as ear swelling and plasma extravasation, suggesting that AVN-C from germinated oats might be a possible therapeutic candidate for mast cell-mediated allergic inflammation [27].

4.3 Antiproliferative ability

AVNs also process an antiproliferative ability, which can inhibit cancer cell development and help balance cell growth [58]. Cell proliferation is the process of cell growth and differentiation. Cancer cells normally grow rapidly and have the potential for tumor development. Vascular smooth muscle cells (VSMCs) are one of the two major factors that lead to atherosclerosis and impaired nitric oxide (NO) bio-availability. Nie et al. examined the effect of AVNs on VSMCs, and the study showed that 120 μM AVNs inhibited VSMC proliferation by more than half (Nie et al., 2006). The same group later investigated the cell-cycle inhibitory mechanism and found that AVN treatment arrested VSMC proliferation in the G1 phase [27].

4.4 Anti-irritant functions

AVNs also process anti-irritant and anti-itching properties. Wang and Eskiw reported that AVN-C reduces H₂O₂-induced oxidative stress by reducing intracellular free radical levels and antioxidant gene transcripts [60]. The expression of gene transcripts encoding proinflammatory cytokines in response to H₂O₂ or tumor necrosis factor-α was significantly decreased after AVN-C treatment. This reduction in cytokine gene transcription occurred concomitantly with reduced phosphorylated NF-κB p65 and decreased NF-κB DNA-binding. AVN-C further induced heme oxygenase-1 (HO-1) expression by increasing Nrf2 DNA-binding activity, demonstrating a second mechanism by which AVN-C attenuates cellular stress. Collectively, these findings indicate that AVN-C protects normal human skin fibroblasts against oxidative stress and inflammatory response through NF-κB inhibition and Nrf2/HO-1 activation. This indicates that the AVNs of oats play an important role in their anti-irritant and anti-itch effects [61,62]

Other potentially interesting biological effects of AVNs include reduced blood pressure, reduced risk of cardiovascular disease, reduced risk of diabetes, and bioavailability. For example, AVNs decrease the release of inflammatory mediators, reducing blood pressure and the risk of cardiovascular disease [63–65]. Additionally, AVNs protect pancreatic β-cells, the insulin-producing cells, from cytokines, resulting in a lower risk of diabetes [12]. These in vitro and in vivo evidences indicated that AVNs involve in reducing free radicals, enhancing activities or transcription of antioxidation enzymes and suppressing proinflammatory cytokines generation, resulting in a number of nutraceutical functions in human health and well-being.

5. Biosynthesis of AVNs

The potential pharmaceutical value of oat AVNs has gradually attracted attentions. However, the cost of extraction, isolation and purification of AVNs from natural oat grains is high, and slows their full development and utilization. AVNs can be extracted using solvent extraction, supercritical CO₂ fluid extraction, and ultrasonication-assisted enzymatic extraction methods. However, when these methods are used, raw materials are easily wasted and solvent contamination occurs [59,66,67] In recent years, modern synthetic biology has promoted the large-scale biomanufacturing of biologically active plant secondary metabolites, greatly enriching the source of such molecules. In this method, identifying key enzymes, characterizing the biosynthetic pathways, then reconstructing the pathway in model plants or microorganisms will accelerate AVNs production and promote the development and utilization of these natural products.

5.1 Biosynthetic pathway of AVNs

The earliest research to determine the biosynthetic pathway of AVNs can be traced back to 20 years ago [68]. Ishihara et al. treated oat leaves with Victoria longumotoxin and found that AVNs could be synthesized in vitro from crude leaf extracts using feruloyl-CoA and 5-hydroxyanthranilate as substrates [69]. Successive studies have defined a type of acyltransferase named hydroxycinnamoyl CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) as the key enzyme for the biosynthesis of AVNs [70]. Later, Susanne et al. firstly studied the concentrations of AVNs and activity of HHT (AsHHT) in dry or steeped, nonmilled or milled, non-heat-treated (raw) or heat-treated oat samples and proved that the concentrations of AVNs and AsHHT activity are positively correlated during the steeping of intact groats at 8 and 20°C [71].

Subsequently, Ren and Wise used benzothiadiazole (BTH) as a resistance activator to induce avenanthramide biosynthesis in the oat cultivar ‘Belle.’ It showed that the application of elicitors increased the activity of HHT in seedling leaves and mature plants, and the magnitude of AVN production in the leaves was higher in the treated plants [28]. Sequence analysis showed that HHT is similar to hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferases (HCT), which is a transferases widely spread in plants, belong to a large family of BAHD acyltransferases that use CoA thioesters as substrates and catalyze the formation of a diverse group of plant metabolites [72,73]. The BAHD members identified to date are all monomeric enzymes with molecular masses ranging from 48 to 55 kDa that share several conserved domains. The first conserved motif is HXXXDG located near the central portion of each enzyme, shared with several other families of acyltransferases that utilize coenzyme A thioesters [73]. The second highly conserved region is the DFGWG motif, located near the carboxyl terminus. Though HCT and HHT belong to the BAHD family and able to catalyze the acylation reactions of several phenolic compounds with p-coumaroyl-CoA, no report indicated the characterized plant HCTs can use 5-hydroxyanthranilic acid as a substrate, whereas oat HHTs mainly prefer 5-hydroxyanthranilic acid as the acyl acceptor [74–76]. These results indicated that HHT is the final enzyme for AVNs accumulation and the substrate specificity of HHT from A. sativa (AsHHT) may play a key role on AVN biosynthesis.

However, the full picture for AVNs biosynthesis remains uncertain and the potential biosynthetic pathway was showed in Figure 4. AVNs are a class of N-aroyl anthranilate alkaloids derived from anthranilic acid that contain an aroyl group attached to a nitrogen atom (N-aroyl). Based on their structures, biosynthesis of AVNs may be a new branch of the phenylpropanoid metabolic pathway for producing hydroxycinnamic acids in oat to adapt environmental stresses [77]. According to the phenylpropanoid metabolic pathway, the process of AVNs biosynthesis begins with the deamination of phenylalanine or tyrosine to form p-coumaric acid. Phenylalanine ammonia lyase (PAL) mainly catalyze the conversion of L-phenylalanine to t-cinnamic acid [78–80]. In this reaction, The PAL active site contains an unusual electrophilic prosthetic group, 4-methylidene-5-one, which acts as a covalent cofactor required for the non-oxidative elimination of ammonia. Consequently, t-cinnamic acid is further condensed with cinnamate 4-hydroxylase (C4H). C4H belongs to the family of cytochrome P450 hydroxylases (P450s), which catalyzes the hydroxylation at the C-4 position of the phenylalanine aromatic ring to form p-coumaric acid [81,82]. In Avena sativa, though genes encoding C4H have not been identified, the activity of C4H could be detected [83]. In addition, p-coumaric acid also can be synthesize from tyrosine via the activity of tyrosine ammonia lyases (TAL) [84,85].

Figure 4. The putative biosynthetic pathway of three major avenanthramides in oat according to [63,68–102].

After that, p-coumaric acid links to acetyl-coenzyme A (CoA) forming p-coumarate-CoA with the catalyzation of p-coumarate-CoA ligase (4CL). 4CLs have been characterized in many plants. They are differentially expressed depending on tissue type and growth stage. In some plants, these genes encode identical or similar proteins (e.g. parsley, loblolly pine, and potato). Structurally divergent isoforms have been identified in other species (e.g. tobacco, A. thaliana, aspen, hybrid poplar, and soybean). These isoenzymes generally display broad but distinct substrate specificities and/or tissue distribution [86–88]. For example, the fourth and final member of the At4CL gene family in A. thaliana, At4CL4, exhibits the rare property of efficiently activating sinapate, besides the usual 4CL substrates (4-coumarate, caffeate, and ferulate), indicating a distinct metabolic function [89]. 4CLs can be classified into two types of proteins (e.g. At4CL1, At4CL2) that are more closely related to each other than to class II proteins (e.g. At4CL3, At4CL4) from the same plant. Class II 4CLs are generally associated with flavonoid biosynthesis, whereas class I 4CLs are closely associated with lignin and other phenylpropanoid biosynthesis [90,91].

Sequentially, AVNs could be further synthesized based on two different pathways. The first pathway is for the hydroxycinnamoyl-based AVN units, where anthranilic acid-based structures (anthranilic acid, 5-hydroxy-anthranilic acid, 4-hydroxy anthranilic acid, 5-hydroxy-4-methoxy anthranilic acid and 4,5-dihydroxy-anthranilic acid) accept an acyl donor by HTT [92]. The parallel pathway produces avenalumic acid-containing AVNs. This route involves condensation of the p-coumaroyl-CoA with malonyl-CoA, producing avenalumoyl-CoA and then synthesize AVN-O and its derivatives by avenalumoyl CoA:3-hydroxylase (AC3’H) and 3-hydroxyavenalumoyl-CoA:O-methyl transferase (HaCoAOMT).

For the following pathway for different AVNs biosynthesis, different studies showed two different potential pathways. The first pathway was reported from Yang et al. [63] Four oat HHT (naming AsHHT1–3 and truncated AsHHT4) were cloned and functional characterized. AsHHT1 expressed in E. coli was found using hydroxyanthranilate and hydroxycinnamoyl-CoAs as co-substrates, indicating that AsHHT1 can synthesize AVN-B, AVN-A, and AVN-L with feruloyl-CoA, 4-coumaroyl-CoA, and avenlumoyl-CoA, respectively. Moreover, the transcripts of AsHHT1 and AsCCoAOMT increased concomitantly under infection stimuli. In contrast, AsHHT4 was consistently expressed in infected and healthy leaves. The constitutive expression of AsHHT4 in healthy oat leaves suggests that this gene may be involved in synthesizing chemical compounds other than phytoalexins. This result showed that three major AVNs (A, B and C) would be catalyzed by HHT with three precursors p-coumaroyl-CoA, caffeoyl-CoA and ferroyl-CoA respectively. And caffeoyl-CoA was synthesized by the function of p-coumaroyl CoA ester 3’-hydroxylase (C3’H) [93,94], whereas ferroyl-CoA was metabolized with caffeoyl-CoA O-methyltransferases (CCoAOMT) using caffeoyl-CoA as substrate [95–99].

Later, the second pathway was revealed [100]. The full-length of AsHHT4 and two new AsHHT genes (AsHHT5 and AsHHT6) from CDC dancers were isolated and AsHHT1 or AsHHT4 in vitro showed the abilities for catalyzing the synthesis of AVN-A and AVN-C. In contrast, their study could not replicate the partial results of Yang et al. in 2004, and purified AsHHT1 or AsHHT4 could not be used as a precursor to synthesize AVN-B. AsHHTs only biosynthesize AVN-A and AVN-C, using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as acyl donors. Instead, an in vitro assay using the purified protein expressed in E. coli in the presence of S-adenosyl methionine showed that purified CCoAOMT from oats (AsCCoAOMT) could convert AVN-C to a product with a retention time and mass spectra identical to those of AVN-B. Therefore, AVN-B can be synthesized via the AsCCoAOMT enzyme through AVN-C methylation. The results elucidated the synthesizing of AVNs might go through an alternative pathway (Figure 4).

Moreover, several evidences showed that the AVNs biosynthesis might go through the second pathway. Michal et al. analyzed the changes in the average contents of the three major AVNs during seed germination (0-196 h). As the result, the total amount of AVNs of three showed a gradually increase in 196 h. Within 48 h, both AVN-A and AVN-C presented the parallel increases of their content, while AVN-B remained low level relative to other two AVNs. However, the content of AVN-C decreased continuously after 48 h, where AVN-B content gradually increased and reached the highest level of three AVNs at 196 h along with the total AVNs content [101]. The similar result also observed in the Wu’s study. As the result, proportion of AVN-B of three AVNs in germination oat seed was significantly increased while the proportion of AVN-C decreased [102]. These results indicated the AVN-A and AVN-C could convert to AVN-B through the second pathway. Therefore, the other avenalumic acid-based AVNs might be synthesized from AVN-O then produce AVN-P using AVN-Q as substrate by the sequential catalyzation of AC3’H and HaCoAOMT (Figure 4). Nevertheless, based on sequencing analysis, at least 72 AsHHT-like proteins have been found in wheat of similar ancestry and the same hexaploid [98], suggesting that oats may have more AsHHT homologous genes. The biological function of the key alkaloid synthetase AsHHT in oats requires further investigation.

5.2 Regulation of AVNs biosynthesis

As previous studies, AVNs biosynthesis requires the co-function with multiple enzymes, therefore identifying the key enzyme for regulating the AVNs production will be important for yielding AVNs in native producers or for metabolic engineering plants or microorganisms. Transcriptional analysis of the genes CCoA3H, CCoAOMT and HHT of oat seeds after anthesis showed that HHT expressed consistently in the whole stage, while CCoA3H and CCoAOMT presented the higher expressions before 18 days after anthesis (DAA) and lower expression after 23 DDA [103]. And the measurement of AVNs content within 30 DDA showed that the total soluble phenol content (SPC, ranging 0.78 to1.09 gGAE/kg d.m.) and total antioxidant capacity (TAC, ranging 13.99 to 18.84 mmol TE/kg d.m.) were also high at around 18–20 DDAs but low after 20 DDA, indicating that the expression level of CCoA3H and CCoAOMT shared high correlation with the accumulation of AVNs [104]. In addition, several studies indicated that PAL also the potential rate-limitation enzyme for AVNs biosynthesis. Wu et al. reported that the highest activity of PAL resulting in the highest level of AVNs in germinated oat [102]. And the PAL upregulated after BHT treatment resulting in a significant enhancement of AVNs level in field oats [105]. Atsushi et al. tested the changes in PAL, C4H and 4CL activities from the oat leaves were treated in penta-N-acetylchitopentaose solution and incubated at 20°C under artificial light, as well as the changes in amounts of AVNs from oat leaves. As the results, amount of AVNs reached the peak at 36 h, while the activity of PAL also presented the highest activity at 36 h, indicating the PAL may involve in AVNs accumulation [83]. Therefore, regulation of these key enzymes activities especially the PAL may play a role on the enhancement of AVNs production.

6. Bioengineering of AVNs by modern biotechnologies

The global demand for nutritional AVNs is increasing owing to public awareness of the potential health benefits of these compounds, such as lower cholesterol levels, suppressed triglyceride accumulation, reduced blood sugar levels, suppressed inflammation, and improved skin health. However, the traditional source of AVNs is oats, and AVN production is unsustainable owing to the various effects of the growth season, growth stage, species, and crop management. Therefore, enhancing plant AVN levels or engineering AVN production in microbes is an attractive alternative for the supply.

6.1 Methods for enhancing AVNs production in oat

As AVNs are secondary metabolites in plants, many factors regulate their accumulation, including gene regulation, environmental stress, elicitors, and growth stages. Many researchers have attempted to produce additional AVNs in oats (Table 3). For example, de Bruijn et al. analyzed the types and levels of AVNs during oat germination. As a result, 28 unique AVNs were annotated in the oat seed extracts, and the AVN content increased by 25 times from seed to seedling, indicating that they could accumulate significantly during germination [49]. The germinating oats treated with methyl jasmonate (MeJA) or abscisic acid (ABA) could yield a 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increase in the AVN content relative to untreated controls (232.6 mg/kg), respectively [106]. Later, to increase the proportion of AVN-C in total AVNs, single or combined treatments with ABA and ascorbic acid (Asc) during the steeping or germination period was performed [101]. Compared to the control (541 μg/g dry weight), the 300 μM-ABA treatment during the steeping period significantly increased the proportion of AVN-C in total AVN (1.8- fold). The content of total AVNs and the proportion of AVN-C within increased significantly (3.35-fold and by 9.28%, respectively) compared to the control when successively treated with 300 μM ABA during the steeping period and 20 mM Asc during the germination period l. Ding et al. evaluated the effects of ultrasound power (25 kHz) on the nutritional properties of germinated oats and the microstructure of oat groats after treatment. The physicochemical properties of oats, including GABA, free sugars, AVNs, total 20 phenolic content, and antioxidant capacity, are enhanced after germination. Levels of AVN-A, AVN-B, AVN-C, and their combined total AVN content reach peak values after 48 h of germination; levels of AVN-C, A, and B are 42.7, 25.9, and 18.6 times higher than those in raw oats [107]. These combined results indicate that changes in plant metabolism can induce AVN accumulation.

Table 3. Summary for methods for enhancing AVNs production in oat.

Factors	Treatment	AVNs yield	Folds of increase	References
Germination	Germination	2.5 µmol/g	25	[49]
	Germinating oats with MeJA or ABA	642.9 mg/kg	2.8	[106]
	Germinating oats with ABA and Asc	1038.07 μg/g DW	3.5	[102]
	Germination	3433.5 μg GAE/mg	3.2	[107]
Elicitors	Partially deacetylated chitin			[108]
	Benzothiadiazole (BTH)	72.7 mg/kg	3.0	[109]
	Oligo-N-acetylchitooligosaccharides	533 µg/g	2.5	[110]
	Calcium ionophore A23187	500 µg/g	2.2	[111]
Expression of related genes	Expression of CBF3 genes with 250 mmol/L NaCl	354.5 mg/kg	1.8–4.6	[112]
Mutagenesis	Ethyl methanesulfonate mutagenesis	227.5 µg/g	2.9	[113]

In addition, many elicitors, such as chitin, BTH, oligo-N-acetylchitooligosaccharides, victorin C, and the calcium ionophore A23187, can activate the biosynthesis of AVNs in oats (Table 4) [109–111]. In 2004, AVN metabolism was investigated using low-molecular-weight partially deacetylated chitin as an elicitor. When oat leaf segments floated on the elicitor solution, AVNs accumulated in the solution. Transferring the elicited oat leaves to solutions containing stable isotope-labeled AVNs rapidly decreased labeled AVNs, suggesting AVN metabolism [108]. BTH are synthetic resistance-inducing chemicals. After treatment with BTH, the relative differences in AVN levels in the BTH-treated vs. non-treated grains were not as apparent as in the leaf tissue [28]. Nevertheless, the BTH-treated grains had a higher mean total AVN content than the untreated controls in all cases. In particular, AVN-A reached 20 mg/g in the Kame cultivar, and the total AVNs reached 72.7 mg/g with BTH treatment.

Table 4. Selected metabolic engineerings of the AVNs-biosynthetic pathways in microorganisms.

Host species	Genes used	Types of AVNs	Yields		References
Saccharomyces cerevisiae	tobacco 4CL and globe artichoke HCT	N-(E)-p-coumaroyl-3- hydroxyanthranilic acid	not mentioned		[74]
Escherichia coli	HCBT from Dianthus caryophyllusenabled and 4CL1from Arabidopsis thaliana	Avn D Avn F	5802 μM 0.54 μM		[114]
Saccharomyces cerevisiae	4cl-2 from tobacco and hct from globe artichoke	YAvnI and YAvnII	120 mg/L		[56,115]
Escherichia coli	TAL, 4CL, HCBT, trpEG In trpD deletion mutant	AVN-D	AVN-D	317.2 mg/L	[116]
		HpaBC SOMT9 and feeding 1121.5 Μm AVN-D	AVN-E	49.5 mg/L
		HapBC and feeding 1121.5 Μm AVN-D	AVN-F	242.0 mg/L
	TAL, 4CL, and HCBT	Supply with p-coumaric acid and 5-hydroxyanthranilate	AVN-A	70.1 mg/L
		Supply with p-coumaric acid and 4-hydroxyanthranilate	AVN-G	89.6 mg/L
	TAL, 4CL, and HCBT, HpaaBC	Feeding 100 μM AVN-A	AVN-B	5.4 μM
		Feeding 100 μM AVN-A	AVN-C	31.1 μM

Ishihara et al. treated oat leaves with oligo-N-acetylchitooligosaccharides [83]. The amount of AVN-A, the major oat phytoalexin, reaches a maximum level of 350 µg/g at 48 h after elicitor treatment, approximately two times higher than that in the untreated oats. In addition, Ishihara et al. treated oats with the calcium ionophore A23187 and analyzed the activation function of this elicitor. Similar to previous results, the calcium ionophore A23187 strongly induces AVNs accumulation. The concentration of AVN-A, a major phytoalexin in oats, is 2.2 times that of the untreated group [111]. These results showed that elicitors activate de-novo AVN synthesis. Furthermore, Kim et al. treated germinating oats with MeJA or ABA, yielding 2.5-fold (582.9 mg/kg FW) and 2.8-fold (642.9 mg/kg FW) increases in the AVNs content, respectively, relative to untreated controls (232.6 mg/kg FW) [106].

Moreover, overexpressing some key genes can also induce AVNs accumulation. Oraby et al. investigated the changes in AVN-A, AVN-B, and AVN-C concentrations in four salinity-tolerant transgenic oat plants containing the CBF3 gene and non-transgenic control plants exposed to different levels of salinity stress [112]. After exposure to 250 mmol/L NaCl, AVN-C drastically increased by 170.9%, 580%, 353.6%, 457.6%, and 229.1% in the control and four transgenic lines, respectively. Among the transgenic lines, Agrogle-1 maintained the highest AVNs concentration at all salinity levels with a maximum of 71.5 mg/kg for AVN-A, 221.0 mg/kg for AVN-C, and 62.0 mg/kg for AVN-B at 250 mmol/L NaCl. These results indicated that overexpressing a stress-tolerance gene (CBF3) increases AVNs production.

Mutagenesis of the original cultivar would change the enzymes or regulators for AVNs biosynthesis intending to enhancing AVNs contents in oat. Oswaldo et al. analyzed the AVNs content from a mutagenized oat population, produced by ethyl methanesulfonate mutagenesis by HPLC-MS² and HPLC equipped with an on-line ABTS⁺ antioxidant detection system. Results showed the qualitative and quantitative differences in the synthesis of AVNs in the different lines, with a total AVNs concentration up to 227.5 µg/g oat seed flour in the highest line, compared with 78.2 µg/g seed in the commercial line. The findings indicated the potential method for developing the oat mutagenized lines with a high concentration of total and/or in- dividual AVNs in the oat seed grain [113].

6.2 Methods for producing AVNs in heterologous microorganisms

Microorganisms are potential platforms for producing a wide range of natural products, including AVNs. The biosynthetic pathway for AVNs has been gradually characterized, facilitating metabolic engineering strategies to produce AVNs in microorganisms (Table 2). AVNs have been successfully bioengineered using various microorganisms, including E. coli and S. cerevisiae. Phenolic esters were produced in S. cerevisiae by coexpressing tobacco 4CL and globe artichoke HCT [74]. This compound is an amide condensation product of p-coumaric acid and 3-hydroxyanthranilic acid and is unexpectedly recruited from yeast metabolism via the HCT enzyme; N-(E)-p-coumaroyl-3-hydroxyanthranilic acid is previously undescribed and shows structural similarity to AVNs. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid reduces the levels of intracellular reactive oxygen species, indicating the potential therapeutic value of this novel compound.

Two years later, Eudes et al. demonstrated in S. cerevisiae that the coexpression of 4-coumarate-CoA ligase (Nt4CL) from A. thaliana and hydroxycinnamoyl/benzoyl-CoA/HCBT from Dianthus caryophyllus produced several cinnamoyl anthranilates when fed anthranilate and various cinnamates [114]. The coexpression of HCBT and Nt4CL1 from tobacco in the E. coli anthranilate-accumulating strain W3110 trpD9923 produces AVN D [N-(4’-hydroxycinnamoyl)-anthranilic acid] and AVN F [N-(3,’4’-dihydroxycinnamoyl)-anthranilic acid] when fed with p-coumarate and caffeate, respectively. Moreover, additional expression in this strain of a tyrosine ammonia-lyase from Rhodotorula glutinis (RgTAL) converted endogenous tyrosine into p-coumarate, producing AVN D from glucose. A 135-fold improvement in AVN D titer was achieved by boosting tyrosine production using two plasmids expressing the 11 genes necessary for tyrosine synthesis from erythrose 4-phosphate and phosphoenolpyruvate. Finally, the expression of either the p-coumarate 3-hydroxylase Sam5 from Saccharothrix espanensis or the hydroxylase complex HpaBC from E. coli endogenously produced caffeate and biosynthesized AVN F. In this application, the maximum theoretical yield for AVN D and AVN F was 27.3 μM and 5.8 mM, respectively [114].

Moglia et al. in 2015 engineered an S. cerevisiae strain with two plant genes (4 cl-2 from tobacco and hct from globe artichoke), producing two novel phenolic compounds N-(E)-p-coumaroyl-3-hydroxyanthranilic acid (yeast avenanthramide I, Yav I) and N-(E)-caffeoyl-3-hydroxyanthranilic acid (yeast avenanthramide II, Yav II)) [115]. By developing a fermentation process for the engineered S. cerevisiae strain, high yields of YavI and YavII II were obtained. Yeast AVN production (YavI and YAv II) reached 120 mg/L after 96 h of fermentation. Yeast AVNs show potential antioxidant and antiproliferative properties in widely used cell models.

Three years later, Finetti et al. evaluated the ability of two novel phenolic compounds (YAVNI and YAVNII) from an engineered strain to inhibit the major hallmarks of colon cancer, including sustained proliferation, migration, and epithelial-mesenchymal transition, using the human colon adenocarcinoma cell line HT29 [56]. Compared to the natural AVNs, both YAVNs and AVNs could inhibit colon cancer cell growth by increasing the expression levels of p21, p27, and p53 proteins. However, YAVNs were more effective than natural compounds at inhibiting cancer cell migration and reversing the major molecular features of the epithelial-mesenchymal transition process, including downregulating E-cadherin mRNA and protein levels.

Lee et al. synthesized nine AVNs from E. coli [116]. They first synthesized AVN D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4CL, HCBT, or anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of AVN-D was produced in vivo. AVN-E and AVN-F were then synthesized from AVN-D using either E. coli harboring HpaBC and SOMT9 or HapBC alone, respectively. AVN-A and AVN-G were synthesized by feeding 5-hydroxyanthranilate or 4- hydroxy-anthranilate to E. coli harboring TAL, 4CL, or HCBT. AVN-B, AVN-C, AVN-H, and AVN-K were synthesized from AVN-A and AVN-G using the same approach as that employed to synthesize AVN-E and AVN-F from AVN-D.

In summary, the introduction of heterologous genes for AVN biosynthesis into several model microbes, including E.coli and S. cerevisiae, produced a series of AVNs. However, none of the engineered microorganisms could de novo synthesize AVN-A, AVN-B, and AVN-C, possibly due to several factors, such as the expression levels of multiple genes, the influence of endogenous genes from the host strain, and differences in codon usage between host strains and plants.

7. Future perspectives

With the awareness of the health benefits of AVNs and the food industry, the market demand for these functional compounds in human food and animal feed will increase. Currently, oats are the main source of AVNs; however, oat production faces many challenges and sustainability issues.

Despite the progress made in AVNs bioengineering using plant elicitors, microorganisms, and transgenic plants, several challenges remain. AVNs are secondary metabolites in oats, and their accumulation and production can be affected by many unpredictable factors, including the levels of target gene regulators, growth stress, and the seasons. Moreover, the AVN biosynthetic pathway shares an upstream pathway with other plant phenylpropanoids. The substrates of the special enzymes involved in AVNs biosynthesis are recognized and combined with different enzymes from competitive pathways. Therefore, the final production of AVNs is regulated by the precursor levels and substrate accumulation. In addition, although natural oat AVNs production can be activated by environmental stress, the yield of other gain components, such as proteins, starch, and lipids, is reduced. Thus, this is neither a sustainable nor economical method for AVNs production. Therefore, an alternative, low-cost, and maneuverable method for AVN production is required.

Bioengineering AVNs using microorganisms is a promising approach for sustainably producing these natural compounds. Thus, enhancing plant AVNs production or engineering biosynthetic pathways in microorganisms will be important in meeting market demands, as the source is more sustainable, environmentally friendly, and safe. Although significant progress has been made, many challenges remain unaddressed. Therefore, to effectively produce AVNs in heterologous systems, efforts should be made to increase the size of the initial precursor pool for use by catalytic enzymes. In addition, little is known about the inducibility of the HHT enzyme(s) and the regulation of carbon flux through precursor pathways in support of AVN biosynthesis. More information and knowledge are still needed for a full understanding of AVN biosynthesis, and people could produce high-quality and high quantities of AVNs through modern breeding and biotechnological approaches.

Abbreviations

AVN	=	avenanthramide
ABA	=	bascisic acid
BTH	=	benzothiadiazole
CCoAOMT	=	caffeoyl-CoA O-methyltransferases
CoA	=	coenzyme A
C4H	=	Cinnamate 4-hydroxylase
C3’H	=	p-coumaroyl CoA ester 3’-hydroxylase
4CL	=	4-coumarate-CoA ligase
DPPH	=	2,2-diphenyl-1-picrylhy-drazyl
HO-1	=	oxygenase-1
HHT	=	hydroxyanthranilateN-hydroxycinnamoyl transferase
HCBT	=	hydroxycinnamoyl/benzoyl-CoA/anthranilate N-hydroxycinnamoyl/benzoyltransferase
L-Phe	=	l-phenylalanine
MeJA	=	methyl jasmonate
N-aroyl	=	nitrogen atom
NF-κ B	=	nuclear factor-kappa B
NO	=	nitric oxide
PAL	=	phenylalanine ammonia lyase
P450	=	P450 hydroxylases
Rg	=	Rhodotorula glutinis
TAL	=	tyrosine ammonia lyase
trpEG	=	anthranilate synthase
VSMC	=	Vascular smooth muscle cell

Author contributions

Xi Xie: Conceptualization, Writing – original draft, Funding acquisition; Miaoyan Lin: Design – original draft, Data Acquisition; Gengsheng Xiao: Conceptualization; Huifan Liu: Data Acquisition; Dongjie Liu: Investigation; Feng Wang: Validation; Lukai Ma: Reviewing – original draft; Qin Wang: Approval of the Final Version; Zhiyong Li: Conceptualization, Review, Editing, Supervision and Funding acquisition.

Availability of data statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the [Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes] under Grant [2019KSYS006]; [Heyuan Sci-tech Commissioner Program] under Grant [220318121670507]; and [Provincial Innovation and Entrepreneurship Training Program for College Students] under Grant [S202211347055].