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.

Newly designed CHS genic primers for four Zingiberaceae species (Alpinia mutica, Alpinia rafflesiana, Hornstedtia leonurus and Scaphochlamys kunstleri)

Flavonoids are secondary metabolites. To date, 2000 naturally occurring flavonoids are known present in plants. These diverse groups of antioxidants are abundant in rhizomes and leaves of Zingiberaceae species. Flavonoids are expressed by many genes. The most studied is chalcone synthase (CHS) gene. However, no study has been performed to study CHS gene in four endemic and pharmacological known Zingiberaceae species: Alpinia mutica, Alpinia rafflesiana, Hornstedtia leonurus and Scaphochlamys kunstleri. Furthermore, A. rafflesiana and S. kunstleri are threatened species. Thus, this study aimed to develop new CHS primers for these selected species. A total of 43 sequences of CHS genes belong to Zingiberaceae and Costaceae were retrieved from the NCBI database. Then, BLASTN was used to check sequence similarities of the retrieved CHS sequences to these four studied species, other Zingiberaceae and Costaceae. In follow, all redundant sequences were excluded and 15 sequences were saved as the final dataset. These 15 sequences were used to design genic primers using Primer3 software and in-silico analysed using OligoAnalyzer™ Tool. This study had successfully designed 12 new CHS genic primers. All the primers can be used for future studies to determine the presence and expression of CHS gene in these four species.

Expression of the ANS, CHS and DFR genes involved in the biosynthesis of anthocyanins in Vaccinium floribundum Kunth from Ecuador, using RT-qPCR.

Vaccinium floribundum Kunth, a wild native species of berry in Ecuador, presents a lot of phenolic compounds, specifically anthocyanins; hence it is considered a natural nutraceutical due to all its nutritional properties. The comparison of the expression of genes is involved in the biosynthesis pathway of anthocyanin of several populations. The aim of the research was to analyze the expression levels of three genes involved in the biosynthesis of anthocyanin in this species collected in two areas of the province of Pichincha: Machachi population of the Mejía canton, with geographic coordinates 0 ° 31′04.8 ″ S 78 ° 37′07.4 ″ W and altitude 3200 meters above sea level, and Pintag population of the Quito cantón, with geographic coordinates 0 ° 24′00.0 ″ S 78 ° 24′00.0 ″ W and altitude 3000 meters above sea level. The gene expression analysis was performed using the quantitative polymerase chain reaction technique and reverse transcription (RT-qPCR). For the population of Machachi, the glyceraldehyde-3-phosphate dehydrogenase gene had an average concentration of 648.59 ng/µL, followed by the chalcone synthase gene with 143.71 ng/µL, then by the dihydroflavonol 4-reductase gene with 59.58 ng/µL and finally by the anthocyanin synthase gene with 39 ng/µL. For the population of Pintag, the glyceraldehyde-3-phosphate dehydrogenase gene has an average concentration of 667.32 ng/µL, followed by the chalcone synthase gene with 157.22 ng/µL, then by the dihydroflavonol 4-reductase gene with 60.42 ng/µL, and finally by the anthocyanin synthase gene with 44.40 ng/µL. Each gene has a similar expression level in both populations, but there are differences when comparing the expression level among genes. Many enzymes, structural genes, and regulatory elements have been observed as transcription factors involved in anthocyanin biosynthesis.

Phylogenetic Implications and Functional Disparity in the Chalcone synthase Gene Family of Common Seagrass Zostera marina

Chalcone synthase (CHS) family are plant type III polyketide synthases that participate in the flavonoid synthesis pathway to induce plant resistance to various biotic and abiotic stresses. Zostera marina, a common seagrass, migrated to terrestrial conditions and returned to the sea, achieving the most severe habitat shift of flowering plants. Given the special evolutionary process, we conducted genome-wide, expression and enzyme activity analyses of the ZosmaCHS family to understand its phylogenetic implications. Various duplication modes led to the expansion of 11 CHS homologs in Z. marina. Based on the phylogenetic relationships, ZosmaCHSs were classified into three clades. Further quantitative real time-PCR analyses of the ZosmaCHS homologs showed different light responses and tissue-specific expression, indicating functional diversification of the ZosmaCHSs. Moreover, the ZosmaCHS proteins clustering with the validated chalcone synthases were recombined into prokaryotic expression systems. All the recombinant proteins showed CHS activity to generate naringenin chalcone with varying catalytic efficiencies. ZosmaCHS07 was regarded as the dominant CHS because of its significant light response and the higher catalytic efficiency. Taken together, the disparity of the expression and enzyme activity indicated that sub-functionalization is the primary mechanism of the expansion of the ZosmaCHSs family.

Transcriptomic and Coexpression Network Analyses Revealed Pine Chalcone Synthase Genes Associated with Pine Wood Nematode Infection

Pine wood nematode (PWN) causes serious diseases in conifers, especially pine species. To investigate the transcriptomic profiles of genes involved in pine-PWN interactions, two different pine species, namely, Pinus thunbergii and P. massoniana, were selected for this study. Weighted gene coexpression network analysis (WGCNA) was used to determine the relationship between changes in gene expression and the PWN population after PWN infection. PWN infection negatively affects the expression of most genes in pine trees, including plant defense-related genes such as genes related to plant hormone signal transduction, plant-pathogen interactions, and the MAPK signaling pathway in plants. However, the expression of chalcone synthase genes and their related genes were proportional to the changes in nematode populations, and chalcone synthase genes were dominant within the coexpression module enriched by genes highly correlated with the nematode population. Many genes that were closely related to chalcone synthase genes in the module were related to flavonoid biosynthesis, flavone and flavonol biosynthesis, and phenylpropanoid biosynthesis. Pine trees could actively adjust their defense strategies in response to changes in the number of invasive PWNs, but the sustained expression of chalcone synthase genes should play an important role in the inhibition of PWN infection.

Study on characterization of chalcone synthase gene from \(\textit{Pueraria lobata}\) and \(\textit{Pueraria mirifica}\) in Vietnam

Two species of genus Pueraria ((Pueraria lobata (synonym: Pueraria montana var. lobata) and Pueraria mirifica (synonym: Pueraria candollei var. mirifica)) are traditional plants used in medicine since ancient times. These plants have been used and became commercially crucial indigenous medicinal plants. Currently, both roots and flowers of P. mirifica are used as a dietary supplement and functional food for women because of their rich source of phytoestrogen and nutrition. However, little information of genes on both species of Pueraria genus (P. lobata and P. mirifica) are known in Vietnam. The purpose of this research is to support more understanding about Chalcone synthase (CHS) genes by determining and sequence analyzing an encoding region of CHS genes that were isolated from P. lobata and P. mirifica. The full-length open reading frame (ORF) sequence CHS was identified with 1170 bp which encodes 389 amino acids by Sanger sequencing. The isolated CHS gene of P. lobata has no difference in sequence with CHS reported on GenBank (D10223.1), whereas a difference of 26 nucleotide positions in CHS sequence of P. mirifica compared with the published gene sequence (JQ409456.1) as consequential having 97.78% genetic similarity. The CHS genes sequence of P. lobata and P. mirifica are homologous with 98.4% because of having 19 nucleotide differences. Chalcone-stilbene synthase N-C terminal, PLN03173, CHS-like, BH0617, fabH are some important domains predicting the CHS genes. Especially, the family signature ‘GVLFGFGPGLTI’ motif of CHS gene as a part of the active-site scaffold contributes to decide the product of cyclization reactions performing the stereochemistry of cyclization which was also observed in P. lobata and P. mirifica, but it was not included for all members in Fabaceae family. With in sillico analysis, the P. lobata and P. mirifica CHS sequences have highly conserved regions to maintain their structure and function, so that it needs further studies to clarify these points.

Multi-tissue transcriptome analysis of two Begonia species reveals dynamic patterns of evolution in the chalcone synthase gene family

Begonia is an important horticultural plant group, as well as one of the most speciose Angiosperm genera, with over 2000 described species. Genus wide studies of genome size have shown that Begonia has a highly variable genome size, and analysis of paralog pairs has previously suggested that Begonia underwent a whole genome duplication. We address the contribution of gene duplication to the generation of diversity in Begonia using a multi-tissue RNA-seq approach. We chose to focus on chalcone synthase (CHS), a gene family having been shown to be involved in biotic and abiotic stress responses in other plant species, in particular its importance in maximising the use of variable light levels in tropical plants. We used RNA-seq to sample six tissues across two closely related but ecologically and morphologically divergent species, Begonia conchifolia and B. plebeja, yielding 17,012 and 19,969 annotated unigenes respectively. We identified the chalcone synthase gene family members in our Begonia study species, as well as in Hillebrandia sandwicensis, the monotypic sister genus to Begonia, Cucumis sativus, Arabidopsis thaliana, and Zea mays. Phylogenetic analysis suggested the CHS gene family has high duplicate turnover, all members of CHS identified in Begonia arising recently, after the divergence of Begonia and Cucumis. Expression profiles were similar within orthologous pairs, but we saw high inter-ortholog expression variation. Sequence analysis showed relaxed selective constraints on some ortholog pairs, with substitutions at conserved sites. Evidence of pseudogenisation and species specific duplication indicate that lineage specific differences are already beginning to accumulate since the divergence of our study species. We conclude that there is evidence for a role of gene duplication in generating diversity through sequence and expression divergence in Begonia.

Salicylic and Methyl Salicylic Acid Affect Quality and Phenolic Profile of Apple Fruits Three Weeks before the Harvest

Effects of spraying over apple trees (Malus domestica; 'Topaz') with methyl salicylic acid (MeSA) and SA during fruit maturation were investigated for quality parameters (weight, firmness, hue angle, red blush, yield) and phenolic profile of the peel and pulp (HPLC–mass spectrometry). These treatment effects were also investigated for activities of the phenylpropanoid pathway enzymes phenylalanine ammonia-lyase, chalcone synthase and isomerase (combined), and flavanone-3β-hydroxylase. The MeSA and SA treatments resulted in poor fruit peel coloration, with higher hue angles and 20% and 10% lower red blush, respectively. Anthocyanin levels were also significantly lower (56%) for MeSA treatment. MeSA stimulated activities of phenylalanine ammonia-lyase and chalcone synthase/isomerase, which resulted in higher levels of flavanols (to 34%), flavonols (to 33%), and hydroxycinnamic acids (to 29%), versus control. Therefore, while these salicylate treatments improve levels of some beneficial polyphenols, they also have negative effects on the external quality characteristics of the fruit.