Comparative Molecular Mechanisms of Biosynthesis of Naringenin and Related Chalcones in Actinobacteria and Plants: Relevance for the Obtention of Potent Bioactive Metabolites

Naringenin and its glycosylated derivative naringin are flavonoids that are synthesized by the phenylpropanoid pathway in plants. We found that naringenin is also formed by the actinobacterium Streptomyces clavuligerus, a well-known microorganism used to industrially produce clavulanic acid. The production of naringenin in S. clavuligerus involves a chalcone synthase that uses p-coumaric as a starter unit and a P450 monoxygenase, encoded by two adjacent genes (ncs-ncyP). The p-coumaric acid starter unit is formed by a tyrosine ammonia lyase encoded by an unlinked, tal, gene. Deletion and complementation studies demonstrate that these three genes are required for biosynthesis of naringenin in S. clavuligerus. Other actinobacteria chalcone synthases use caffeic acid, ferulic acid, sinapic acid or benzoic acid as starter units in the formation of different antibiotics and antitumor agents. The biosynthesis of naringenin is restricted to a few Streptomycess species and the encoding gene cluster is present also in some Saccharotrix and Kitasatospora species. Phylogenetic comparison of S. clavuligerus naringenin chalcone synthase with homologous proteins of other actinobacteria reveal that this protein is closely related to chalcone synthases that use malonyl-CoA as a starter unit for the formation of red-brown pigment. The function of the core enzymes in the pathway, such as the chalcone synthase and the tyrosine ammonia lyase, is conserved in plants and actinobacteria. However, S. clavuligerus use a P450 monooxygenase proposed to complete the cyclization step of the naringenin chalcone, whereas this reaction in plants is performed by a chalcone isomerase. Comparison of the plant and S. clavuligerus chalcone synthases indicates that they have not been transmitted between these organisms by a recent horizontal gene transfer phenomenon. We provide a comprehensive view of the molecular genetics and biochemistry of chalcone synthases and their impact on the development of antibacterial and antitumor compounds. These advances allow new bioactive compounds to be obtained using combinatorial strategies. In addition, processes of heterologous expression and bioconversion for the production of naringenin and naringenin-derived compounds in yeasts are described.

Biosynthesis of ethyl caffeate via caffeoyl-CoA acyltransferase expression in Escherichia coli

Hydroxycinnamic acids (HCs) are natural compounds that form conjugates with diverse compounds in nature. Ethyl caffeate (EC) is a conjugate of caffeic acid (an HC) and ethanol. It has been found in several plants, including Prunus yedoensis, Polygonum amplexicaule, and Ligularia fischeri. Although it exhibits anticancer, anti-inflammatory, and antifibrotic activities, its biosynthetic pathway in plants still remains unknown. This study aimed to design an EC synthesis pathway and clone genes relevant to the same. Genes involved in the caffeic acid synthesis pathway (tyrosine ammonia-lyase (TAL) and p-coumaric acid hydroxylase (HpaBC)) were introduced into Escherichia coli along with 4-coumaroyl CoA ligase (4CL) and acyltransferases (AtCAT) cloned from Arabidopsis thaliana. In presence of ethanol, E. coli harboring the above genes successfully synthesized EC. Providing more tyrosine through the overexpression of shikimate-pathway gene-module construct and using E. coli mutant enhanced EC yield; approximately 116.7 mg/L EC could be synthesized in the process. Synthesis of four more alkyl caffeates was confirmed in this study; these might potentially possess novel biological properties, which would require further investigation.

Photosynthetic apparatus efficiency, phenolic acid profiling and pattern of chosen phytohormones in pseudometallophyte Alyssum montanum

The present study investigated the response of non-metallicolous (NM) and metallicolous (M) Alyssum montanum shoots cultured in vitro on a medium supplemented simultaneously with heavy metals (HMs) to identify mechanisms involved in alleviating metal-induced damage. Plant status in respect to photosynthetic apparatus efficiency was determined and linked with changes in biochemical composition of shoots, namely phenolic acids’ and stress-related phytohormones. Results showed the considerable inter-ecotype differences in (1) the photosynthetic pigments’ amount, (2) the functioning of membrane electron transporters as well as (3) the linear and alternative electron transport pathways, whose lower values were reported in NM than in M HM-treated culture. Photosynthetic apparatus protection in M specimens was assured by the activation of cinnamic acid synthesis (by phenylalanine ammonia lyase) and its further transformations to benzoic acid derivatives with high ability to counteract oxidative stress, that was accompanied by the overexpression of jasmonic acid stimulating antioxidant machinery. In turn, detrimental HM effects on NM shoots could result from the diminution of most phenolics’ accumulation, and only the content of coumarate (produced by bifunctional phenylalanine/tyrosine ammonia lyase) and rosmarinic acid increased. All these together with an enhanced concentration of abscisic acid might suggest that NM strategy to cope with HMs is based mostly on a restriction of metal movement with transpiration flow and their limited distribution in leaves. Summarizing, our findings for the first time point out the physiological and metabolic adaptation of pseudometallophyte A. montanum to adverse conditions.

Biosynthesis of resveratrol using metabolically engineered Escherichia coli

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a phenolic compound widely used in pharmaceutics and nutraceutics. Although resveratrol is produced by some plant species, including grapes, peanuts, and berries, the content of resveratrol and its derivatives are very low. Therefore, an alternative biosynthetic method using microorganisms, such as Escherichia coli, has been developed over the past two decades. In the present study, a resveratrol-over-producing E. coli strain was developed using three strategies. First, we increased the synthesis of p-coumaric acid, a precursor of resveratrol, by manipulating genes in the shikimate pathway of E. coli. Second, three genes involved in resveratrol biosynthesis, such as tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS), were cloned from diverse sources, such as plants and microorganisms, and the best combination was selected to maximize resveratrol production in E. coli. Finally, culture conditions, such as cell concentration, culture temperature, and carbon sources, were established for optimal resveratrol production. Through these strategies, approximately 80.4 mg/L of resveratrol was biosynthesized after 48 h of culture using glycerol as a carbon source.

Biochemical changes in ridge gourd (Luffa acutangula l.) cultivars induced by root knot nematodes

Biochemical changes in different ridge gourd cultivars inoculated with Meloidogyne incognita were investigated. All the parameters showed higher activity during post infection period, both at shoots and roots level. Cultivar Priya had significant higher activity of phenylalanine ammonia lyase (PAL) than rest of the cultivars in infected shoots. However, it had only significant higher PAL expression in infected roots of Harsha cultivar of ridge gourd. All the cultivars showed non-significant changes in infected shoots for tyrosine ammonia lyase (TAL) contents. In infected roots, culture BSS 1009 showed significant increase in TAL than cultivars Harsha and Aarti, however Priya only had significant higher expression of TAL than culture Harsha. For peroxidase contains, all the cultivars displayed significant variation at infected shoots and roots level. Highest increase in peroxidase was noticed in resistant than susceptible cultivars in infected shoots of different ridge gourd cultivars. In general, with few exceptions resistant cultivars had higher catalase activity both in infected shoots and roots of different ridge gourd cultivars.

The Mandelate Pathway, an Alternative to the Phenylalanine Ammonia Lyase Pathway for the Synthesis of Benzenoids in Ascomycete Yeasts

ABSTRACT Benzenoid-derived metabolites act as precursors for a wide variety of products involved in essential metabolic roles in eukaryotic cells. They are synthesized in plants and some fungi through the phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) pathways. Ascomycete yeasts and animals both lack the capacity for PAL/TAL pathways, and metabolic reactions leading to benzenoid synthesis in these organisms have remained incompletely known for decades. Here, we show genomic, transcriptomic, and metabolomic evidence that yeasts use a mandelate pathway to synthesize benzenoids, with some similarities to pathways used by bacteria. We conducted feeding experiments using a synthetic fermentation medium that contained either 13C-phenylalanine or 13C-tyrosine, and, using methylbenzoylphosphonate (MBP) to inhibit benzoylformate decarboxylase, we were able to accumulate intracellular intermediates in the yeast Hanseniaspora vineae. To further confirm this pathway, we tested in separate fermentation experiments three mutants with deletions in the key genes putatively proposed to form benzenoids (Saccharomyces cerevisiae aro10Δ, dld1Δ, and dld2Δ strains). Our results elucidate the mechanism of benzenoid synthesis in yeast through phenylpyruvate linked with the mandelate pathway to produce benzyl alcohol and 4-hydroxybenzaldehyde from the aromatic amino acids phenylalanine and tyrosine, as well as sugars. These results provide an explanation for the origin of the benzoquinone ring, 4-hydroxybenzoate, and suggest that Aro10p has benzoylformate and 4-hydroxybenzoylformate decarboxylase functions in yeast. IMPORTANCE We present here evidence of the existence of the mandelate pathway in yeast for the synthesis of benzenoids. The link between phenylpyruvate- and 4-hydroxyphenlypyruvate-derived compounds with the corresponding synthesis of benzaldehydes through benzoylformate decarboxylation is demonstrated. Hanseniaspora vineae was used in these studies because of its capacity to produce benzenoid derivatives at a level 2 orders of magnitude higher than that produced by Saccharomyces. Contrary to what was hypothesized, neither β-oxidation derivatives nor 4-coumaric acid is an intermediate in the synthesis of yeast benzenoids. Our results might offer an answer to the long-standing question of the origin of 4-hydroxybenzoate for the synthesis of Q10 in humans.

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450

BackgroundCaffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far.ResultsThe herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 μM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even an initially unproductive Cytochrome P450/ redox system combination productive.ConclusionsThe caffeic acid titer achieved in this study is about 25% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.