Ocimum kilimandscharicum 4CL11 negatively regulates adventitious root development via accumulation of flavonoid glycosides.

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their in planta functions remain enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11, and -15) in Nicotiana benthamiana. Interestingly, Ok4CL11 overexpression (OE) caused a rootless or reduced root growth phenotype, whereas overexpression of Ok4CL15 produced normal adventitious root (AR) growth. Ok4CL11 overexpression in N. benthamiana resulted in upregulation of genes involved in flavonoid biosynthesis and associated glycosyltransferases accompanied by accumulation of specific flavonoid-glycosides (kaempferol-3-rhamnoside, kaempferol-3,7-O-bis-alpha-l-rhamnoside [K3,7R], and quercetin-3-O-rutinoside) that possibly reduced auxin levels in plants, and such effects were not seen for Ok4CL7 and -15. Docking analysis suggested that auxin transporters (PINs/LAXs) have higher binding affinity to these specific flavonoid-glycosides, and thus could disrupt auxin transport/signaling, which cumulatively resulted in a rootless phenotype. Reduced auxin levels, increased K3,7R in the middle and basal stem sections, and grafting experiments (intra and inter-species) indicated a disruption of auxin transport by K3,7R and its negative effect on AR development. Supplementation of flavonoids and the specific glycosides accumulated by Ok4CL11-OE to the wild-type N. benthamiana explants delayed the AR emergence and also inhibited AR growth. While overexpression of all three Ok4CLs increased lignin accumulation, flavonoids, and their specific glycosides were accumulated only in Ok4CL11-OE lines. In summary, our study reveals unique indirect function of Ok4CL11 to increase specific flavonoids and their glycosides, which are negative regulators of root growth, likely involved in inhibition of auxin transport and signaling.

[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Bibenzyl synthesis in Cannabis sativa L.

This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the 'bibenzyls'. In planta, bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti-inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis, in a three-step sequence. First, various hydroxycinnamic acids are esterified to acyl-coenzyme A (CoA) by a member of the 4-coumarate-CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double-bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro-CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl-CoA with these dihydro-hydroxycinnamoyl-CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this 'bibenzyl synthase' enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non-canonical and more flexible dihydro-hydroxycinnamoyl-CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro. Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing 'parts prospecting' inventory that supports the rational metabolic engineering of natural product therapeutics.

Transcriptomic Analysis Reveals Novel Regulators of the Scots Pine Stilbene Pathway.

Stilbenes accumulate in Scots pine heartwood where they have important roles in protecting wood from decaying fungi. They are also part of active defense responses, and their production is induced by different (a)biotic stressors. The specific transcriptional regulators as well as the enzyme responsible for activating the stilbene precursor cinnamate in the pathway are still unknown. UV-C radiation was the first discovered artificial stress activator of the pathway. Here, we describe a large-scale transcriptomic analysis of pine needles in response to UV-C and treatment with translational inhibitors, both activating the transcription of stilbene pathway genes. We used the data to identify putative candidates for the missing CoA ligase and for pathway regulators. We further showed that the pathway is transcriptionally activated by phosphatase inhibitor, ethylene and jasmonate treatments, as in grapevine, and that the stilbene synthase promoter retains its inducibility in some of the tested conditions in Arabidopsis, a species that normally does not synthesize stilbenes. Shared features between gymnosperm and angiosperm regulation and partially retained inducibility in Arabidopsis suggest that pathway regulation occurs not only via ancient stress-response pathway(s) but also via species-specific regulators. Understanding which genes control the biosynthesis of stilbenes in Scots pine aids breeding of more resistant trees.

Genome-Wide Identification and Expression Analysis of the 4-Coumarate: CoA Ligase Gene Family in Solanum tuberosum.

4-coumarate: CoA ligase (4CL) is not only involved in the biosynthetic processes of flavonoids and lignin in plants but is also closely related to plant tolerance to abiotic stress. UV irradiation can activate the expression of 4CL genes in plants, and the expression of 4CL genes changed significantly in response to different phytohormone treatments. Although the 4CL gene has been cloned in potatoes, there have been fewer related studies of the 4CL gene family on the potato genome-wide scale. In this study, a total of 10 potato 4CL genes were identified in the potato whole genome. Through multiple sequence alignment, phylogenetic analysis as well as gene structure analysis indicated that the potato 4CL gene family could be divided into two subgroups. Combined with promoter cis-acting element analysis, transcriptome data, and RT-qPCR results indicated that potato 4CL gene family was involved in potato response to white light, UV irradiation, ABA treatment, MeJA treatment, and PEG simulated drought stress. Abiotic stresses such as UV, ABA, MeJA, and PEG could promote the up-regulated expression of St4CL6 and St4CL8 but inhibits the expression of St4CL5. The above results will increase our understanding of the evolution and expression regulation of the potato 4CL gene family and provide reference value for further research on the molecular biological mechanism of 4CL participating in response to diverse environmental signals in potatoes.

Systematic Analysis and Expression Profiles of the 4-Coumarate: CoA Ligase (4CL) Gene Family in Pomegranate (Punica granatum L.).

4-Coumarate:CoA ligase (4CL, EC6.2.1.12), located at the end of the phenylpropanoid metabolic pathway, regulates the metabolic direction of phenylpropanoid derivatives and plays a pivotal role in the biosynthesis of flavonoids, lignin, and other secondary metabolites. In order to understand the molecular characteristics and potential biological functions of the 4CL gene family in the pomegranate, a bioinformatics analysis was carried out on the identified 4CLs. In this study, 12 Pg4CLs were identified in the pomegranate genome, which contained two conserved amino acid domains: AMP-binding domain Box I (SSGTTGLPKGV) and Box II (GEICIRG). During the identification, it was found that Pg4CL2 was missing Box II. The gene cloning and sequencing verified that this partial amino acid deletion was caused by genome sequencing and splicing errors, and the gene cloning results corrected the Pg4CL2 sequence information in the 'Taishanhong' genome. According to the phylogenetic tree, Pg4CLs were divided into three subfamilies, and each subfamily had 1, 1, and 10 members, respectively. Analysis of cis-acting elements found that all the upstream sequences of Pg4CLs contained at least one phytohormone response element. An RNA-seq and protein interaction network analysis suggested that Pg4CL5 was highly expressed in different tissues and may participate in lignin synthesis of pomegranate. The expression of Pg4CL in developing pomegranate fruits was analyzed by quantitative real-time PCR (qRT-PCR), and the expression level of Pg4CL2 demonstrated a decreasing trend, similar to the trend of flavonoid content, indicating Pg4CL2 may involve in flavonoid synthesis and pigment accumulation. Pg4CL3, Pg4CL7, Pg4CL8, and Pg4CL10 were almost not expressed or lowly expressed, the expression level of Pg4CL4 was higher in the later stage of fruit development, suggesting that Pg4CL4 played a crucial role in fruit ripening. The expression levels of 4CL genes were significantly different in various fruit development stages. The results laid the foundation for an in-depth analysis of pomegranate 4CL gene functions.

4-Coumarate:coenzyme A ligase isoform 3 from Piper nigrum (Pn4CL3) catalyzes the CoA thioester formation of 3,4-methylenedioxycinnamic and piperic acids.

Black pepper, dried green fruit of Piper nigrum L., is a household spice most popular in the world. Piperine, the pungency compound of black pepper, is proposed to partially arise from phenylpropanoid pathway. In the biosynthesis of piperine, 4-coumarate:CoA ligase (4CLs) must play a pivotal role in activating intermediate acids to corresponding CoA thioesters to serve as substrates. Based on transcriptome data, we isolated three P. nigrum 4CL isoforms (Pn4CL1, -2, and -3) from unripe peppercorn. These Pn4CLs were expressed in E. coli for in vitro enzyme assay with putative substrates, namely cinnamic, coumaric, ferulic, piperonylic, 3,4-methylenedioxycinnamic (3,4-MDCA), and piperic acids. Phylogenetic analysis and substrate usage study indicated that Pn4CL1, active towards coumaric and ferulic acids, belongs to class I 4CL for lignin synthesis. Pn4CL2 was a typical cinnamate-specific coumarate:CoA ligase-like (CLL) protein. The Pn4CL3, as class II enzyme, exhibited general 4CL activity towards coumaric and ferulic acids. However, Pn4CL3 was also active towards piperonylic acid, 3,4-MDCA, and piperic acid. Pn4CL3 possessed ∼2.6 times higher catalytic efficiency (kcat/KM) towards 3,4-MDCA and piperic acid than towards coumaric and ferulic acids, suggesting its specific role in piperine biosynthesis. Different substrate preference among the Pn4CL isoforms can be explained by 3-dimensional protein structure modeling, which demonstrated natural variants in amino acid residues of binding pocket to accommodate different substrates. Quantitative PCR analysis of these isoforms indicated that Pn4CL1 transcript level was highest in the roots whereas Pn4CL2 in the fruits and Pn4CL3 in the leaves.

Construction of synthetic pathways for raspberry ketone production in engineered Escherichia coli.

Raspberry ketone is an important ingredient in the flavor and fragrance industries. Due to its low content in fruits and vegetables, the production of natural raspberry ketone using heterologous synthesis in microbial strains is recently attracting increased attention. In this work, a heterologous pathway to produce raspberry ketone from p-coumaric acid, including 4-coumarate: CoA ligase (4CL), benzalacetone synthase (BAS), and raspberry ketone/zingerone synthase (RZS1) from plants, was successfully assembled in Escherichia coli. When the RZS1 gene was introduced into E. coli and co-expressed with two other genes, the intermediate 4-hydroxybenzylidene acetone in the pathway was almost completely transformed into a raspberry ketone. Substituting TB medium for M9 medium increased raspberry ketone titers by 3-4 times. Furthermore, the heterologous pathway was partitioned into two modules; module one produced p-coumaroyl-CoA from p-coumaric acid by 4CL, and module two produced raspberry ketone from coumaroyl-CoA by the action of BAS and RZS1. Optimizing the balanced expression of the two modules, it was shown that moderate expression of module one and high expression of module two was the best combination to enhance raspberry ketone production. The engineered strain CZ-8 reached 90.97 mg/l of raspberry ketone, which was 12 times higher than previously reported. In addition, the preferred approach of the heterologous pathway was related to the heterologous genes from different sources; for example, 4CL from Arabidopsis thaliana seemed to be more suitable for raspberry ketone production than that from Petroselinum crispum. This work paves an alternative way for future economic production of natural raspberry ketone.

Structural, functional and evolutionary diversity of 4-coumarate-CoA ligase in plants.

MAIN CONCLUSIONS: The 4-coumarate-CoA ligases (4CL) contribute in channelizing flux of different phenylpropanoid biosynthetic pathways. Expression of 4CL is optimized at developmental stages and in response to environmental triggers such as biotic and abiotic stresses. The enzyme is valuable in metabolic pathway engineering for curcuminoids, resveratrol, biofuel production and nutritional improvement. Vigorous analysis of regulation at functional and expression level is obligatory to attain efficient commercial production of candidate metabolites using 4CL. Phenylpropanoid pathway provides precursors for numerous secondary metabolites in plants. In this pathway, 4-coumarate-CoA ligase (EC 6.2.1.12, 4CL) is the main branch point enzyme which generates activated thioesters. Being the last enzyme of three shared common steps in general phenylpropanoid pathway, it contributes to channelize precursors for different phenylpropanoids. In plants, 4CL enzymes are present in multiple isoforms and encoded by small gene family. It belongs to adenylate-forming enzyme family and catalyzes the reaction that converts hydroxy or methoxy cinnamic acid derivatives to corresponding thioesters. These thioesters are further utilized for biosynthesis of phenylpropanoids, which are known for having numerous nutritional and medicinal applications. In addition, the 4CL enzymes have been characterized from various plants for their role in plant physiology or in biotic and abiotic stresses. Furthermore, specific isoforms are differentially regulated upon exposure to diverse stimuli leading to flux diversion toward the particular metabolite biosynthesis. Evolutionary studies showed that 4CL separately evolved after monocot and dicot segregation. Here, we provide a comprehensive review on 4CL, which includes evolution, function, gene/protein structure, role in metabolite biosynthesis and cellular partition, and their regulation. Based on the available data, we have explored the scope for pathway engineering by utilizing 4CL enzymes.

Cloning and Functional Characterization of Two 4-Coumarate: CoA Ligase Genes from Selaginella moellendorffii.

Selaginella is an extant lycopodiophyte genus, which is representative of an ancient lineage of tracheophytes. The important evolutionary status makes it a valuable resource for the study of metabolic evolution in vascular plants. 4-coumarate: CoA ligase (4CL) is the pivotal enzyme that controls the flow of carbon through the phenylpropanoid metabolic pathway into the specific lignin, flavonoid, and wall-bound phenolics biosynthesis pathways. Although 4CLs have been extensively characterized in other vascular plants, little is known of their functions in Selaginella. Here, we isolated two 4CL genes (Sm4CL1 and Sm4CL2) from Selaginella moellendorffii. Based on the enzymatic activities of the recombinant proteins, both of these genes encoded bona fide 4CLs. The 4CL isoforms in S. moellendorffii have different activities: Sm4CL2 was more active than Sm4CL1. The enzymatic properties and gene expression patterns indicated that the 4CL genes have been conserved in the evolution of vascular plants.

Molecular analysis of caffeoyl residues related to pigmentation in green cotton fibers.

The pigment components in green cotton fibers were isolated and identified as 22-O-caffeoyl-22-hydroxymonodocosanoin and 22-O-caffeoyl-22-hydroxydocosanoic acid. The concentration of 22-O-caffeoyl-22-hydroxymonodocosanoin correlated positively with the degree of colour in the green fibers, indicating a role for caffeoyl derivatives in the pigmentation of green cotton fibers. Upland cotton (Gossypium hirsutum L.) contains four genes, Gh4CL1-Gh4CL4, encoding 4-coumarate:CoA ligases (4CLs), key enzymes in the phenylpropanoid biosynthesis pathway. In 15-24-day post-anthesis fibers, the expression level of Gh4CL1 was very low, Gh4CL3 had a similar expression level in both white and green cottons, Gh4CL2 had a significantly higher expression level in green fibers than in white fibers, while Gh4CL4 had a higher expression level in white fibers than in green fibers. According to enzyme kinetics analysis, Gh4CL1 displayed a preference for 4-coumarate, Gh4CL3 and Gh4CL4 exhibited a somewhat low but still prominent activity towards ferulate, while Gh4CL2 had a strong preference for caffeate and ferulate. These results suggest that Gh4CL2 might be involved in the metabolism of caffeoyl residues and related to pigment biosynthesis in green cotton fibers. Our findings provide insights for understanding the biochemical and molecular mechanisms of pigmentation in green cotton fibers.

Synthesis of rosmarinic acid analogues in Escherichia coli.

OBJECTIVES: To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS). RESULTS: Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues. CONCLUSION: This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

Engineered synthesis of rosmarinic acid in Escherichia coli resulting production of a new intermediate, caffeoyl-phenyllactate.

OBJECTIVES: To achieve high production of rosmarinic acid and derivatives in Escherichia coli which are important phenolic acids found in plants, and display diverse biological activities. RESULTS: The synthesis of rosmarinic acid was achieved by feeding caffeic acid and constructing an artificial pathway for 3,4-dihydroxyphenyllactic acid. Genes encoding the following enzymes: rosmarinic acid synthase from Coleus blumei, 4-coumarate: CoA ligase from Arabidopsis thaliana, 4-hydroxyphenyllactate 3-hydroxylase from E. coli and D-lactate dehydrogenase from Lactobacillus pentosus, were overexpressed in an L-tyrosine over-producing E. coli strain. The yield of rosmarinic acid reached ~130 mg l(-1) in the recombinant strain. In addition, a new intermediate, caffeoyl-phenyllactate (~55 mg l(-1)), was also produced by the engineered E. coli strain. CONCLUSION: This work not only leads to high yield production of rosmarinic acid and analogues, but also sheds new light on the construction of the pathway of rosmarinic acid in E. coli.

4-Coumarate-CoA ligase (4-CL) enhances flavonoid accumulation, lignin synthesis, and fruiting body formation in Ganoderma lucidum.

It is now understood that 4-Coumarate-CoA ligases (4-CL) are pivotal in bridging the phenylpropanoid metabolic pathway and the lignin biosynthesis pathway in plants. However, limited information on 4-CL genes and their functions in fungi is available. In this study, we cloned the 4-CL gene (Gl21040) from Ganoderma lucidum, which spans 2178 bp and consists of 10 exons and 9 introns. We also developed RNA interference and overexpression vectors for Gl21040 to investigate its roles in G. lucidum. Our findings indicated that in the Gl21040 interference transformants, 4-CL enzyme activities decreased by 31 %-57 %, flavonoids contents decreased by 10 %-22 %, lignin contents decreased by 20 %-36 % compared to the wild-type (WT) strain. Conversely, in the Gl21040 overexpression transformants, 4-CL enzyme activity increased by 108 %-143 %, flavonoids contents increased by 8 %-37 %, lignin contents improved by 15 %-17 % compared to the WT strain. Furthermore, primordia formation was delayed by approximately 10 days in the Gl21040-interferenced transformants but occurred 3 days earlier in the Gl21040-overexpressed transformants compared to the WT strain. These results underscored the involvement of the Gl21040 gene in flavonoid synthesis, lignin synthesis, and fruiting body formation in G. lucidum.

Global lysine succinylation analysis unveils post-translational regulation effect on phenylpropanoid metabolism remodeling during Lonicera japonica flower development.

Lonicera japonica plays a significant role in traditional Chinese medicine and as a food source, making it a focus of studies on protein succinylation and its potential role in regulating secondary metabolism during flower development. This study aimed to clarify the regulatory mechanism of protein succinylation on phenylpropanoid-related phenotypic changes by conducting a global lysine succinylation proteomic analysis across different flowering stages. A total of 586 lysine succinylated peptides in 303 proteins were identified during early and late floral stages. Functional enrichment analysis revealed that succinylated proteins primarily participated in the tricarboxylic acid (TCA) cycle, amino acid metabolism, and secondary metabolism. The abundance of succinylated aspartate transaminase (AT), 4-coumarate-CoA ligase (4CL), and phenylalanine N-hydroxylase (CYP79A2) in phenylpropanoid metabolism varied during flower development. In vitro experiments demonstrated that succinylation increased AT activity while inhibited 4CL activity. Decreased levels of total flavonoids and phenolic acids indicated significant alterations in phenylpropanoid metabolism during later floral stages. These results suggest that succinylation of TCA cycle proteins not only influences flower development but also, together with AT-4CL-CYP79A2 co-succinylation, redirects phenylpropanoid metabolism during flower development in L. japonica.

Comprehensive genomic identification and expression analysis 4CL gene family in apple.

To clarify the structural characteristics, phylogeny, biological function and regulation of 4-coumarate-CoAligase (4CL) in anthocyanin synthesis, the 4CL gene family members in apples were identified and bioinformatic analysis was performed. qRT-PCR was used to analyze the expression levels of this gene family members in different apple varieties, and the role of the 4CL gene in apple anthocyanin synthesis was preliminaries clarified, which provided a certain theoretical basis for the regulatory network of apple anthocyanin synthesis. The results showed that a total of 69 members of the 4CL gene family were identified in the apple (Malus domestica Brokh.), encoding amino acids ranging from 97 to 2310 with theoretical isoelectric points ranging from 5.28 to 9.84. The 69 4CL family members were distributed on 17 chromosomes in the apple, among which chromosome 17 had the largest distribution (9 members), followed by chromosome 9 (7 members), chromosomes 16 and 14 (6 members each), and chromosomes 15 and 13 (5 members each). The subcellular localization prediction showed that apple 4CL gene family members were mainly expressed in cytoplasm, chloroplast, nucleus and cell membrane, with a small amount of expression in mitochondria, vacuoles, peroxisomes, cytoskeleton, golgi and cell matrix, but not in endoplasmic reticulum. The secondary structures are mainly α-helices and irregular coils. Microarray expression profile analysis showed that the expression levels of each member in apple were related to fruit variety and tissue structure, and the expression levels were mainly higher in fruit, flower and leaf. Real-time PCR analysis showed that the expression level of each member was directly proportional to the degree of fruit coloring and anthocyanin accumulation. The expression levels of Md4CL10 and Md4CL23 in 'Astar' (G4) apple fruit skin with the highest anthocyanin content were 516, 20 and 2 times higher than those in 'Chengji NO.1' (G1), 'Golden Delicious' (G2) and 'Ruixue' (G3), respectively.

Programmed Cell Death May Be Involved in the Seedless Phenotype Formation of Oil Palm.

Oil palm (Elaeis guineensis Jacq.) is a well-known vegetable oil-yielding crop. Seedlessness is one of the most prominent traits in oil palm due to its low processing costs and high oil content. Nevertheless, an extensive study on molecular mechanisms regulating seedless phenotype formation in oil palm is very limited so far. In this study, stigma, style, and ovary from seedless and seeded (Tenera and Pisifera) oil palm trees were used to investigate the possible mechanism. Results showed that non-pollination resulted in no fruits, and self- and cross-pollinations resulted in seedless fruits, while boron treatment had no effect on seedless phenotype formation, implying that seedless trees have incomplete self and outcrossing incompatibility. Furthermore, the transcriptome data analysis highlighted eight programmed cell death (PCD) genes and three groups of PCD-related genes: 4-coumarate-CoA ligase (4CL), S-RNase, and MADS-box. The majority of these genes were significantly up-regulated in the stigma and style of Seedless palm trees compared to Tenera and Pisifera. In addition, the co-expression network analysis confirmed the significant correlation among these genes. Moreover, two simple sequence repeats (SSR) markers (S41 and S44) were developed to identify the seedless phenotype. The up-regulation of 4CL and MADS-box TFs activated the expression of PCD genes; on the other hand, S-RNase resulted in pollen tube RNA degradation and triggered PCD. While the link between PCD and seedless phenotype formation in oil palm has not been extensively studied to date, these findings suggest a role of PCD in pollen tube lethality, leading to double fertilization failure and the seedless phenotype.

Efficient biosynthesis of pinosylvin from lignin-derived cinnamic acid by metabolic engineering of Escherichia coli.

BACKGROUND: The conversion of lignin-derived aromatic monomers into valuable chemicals has promising potential to improve the economic competitiveness of biomass biorefineries. Pinosylvin is an attractive pharmaceutical with multiple promising biological activities. RESULTS: Herein, Escherichia coli was engineered to convert the lignin-derived standard model monomer cinnamic acid into pinosylvin by introducing two novel enzymes from the wood plant: stilbene synthase from Pinus pinea (PpSTS) and 4-Coumarate-CoA ligase from Populus trichocarpa (Ptr4CL4). The expression of Ptr4CL4 drastically improved the production of pinosylvin (42.5 ± 1.1 mg/L), achieving values 15.7-fold higher than that of Ptr4CL5 (another 4-Coumarate-CoA ligase from Populus trichocarpa) in the absence of cerulenin. By adjusting the expression strategy, the optimized engineered strain produced pinosylvin at 153.7 ± 2.2 mg/L with an extremely high yield of 1.20 ± 0.02 mg/mg cinnamic acid in the presence of cerulenin, which is 83.9% ± 1.17 of the theoretical yield. This is the highest reported pinosylvin yield directly from cinnamic acid to date. CONCLUSION: Our work highlights the feasibility of microbial production of pinosylvin from cinnamic acid and paves the way for converting lignin-related aromatics to valuable chemicals.

Alleviation of the Byproducts Formation Enables Highly Efficient Biosynthesis of Rosmarinic Acid in Saccharomyces cerevisiae.

Rosmarinic acid as a polyphenolic compound has great values in the pharmaceutical, cosmetic, and food industries. To achieve efficient biosynthesis of rosmarinic acid, the major obstacles such as imbalanced metabolic flux among branching pathways and substrate promiscuity of pathway enzymes should be eliminated. Here, a rosmarinic acid producing Saccharomyces cerevisiae strain was constructed by introducing codon optimized d-lactate dehydrogenase gene mutant (OD-LDHY52A), 4-coumarate CoA ligase gene (OPc4CL2), and rosmarinic acid synthase gene (OMoRAS) into a previously constructed caffeic acid hyper-producer. To identify the metabolic bottleneck, the substrate specificity of OPc4CL2 and OMoRAS was figured out by bioconversion experiments and HPLC-MS/MS analysis. Subsequently, the byproducts formation was alleviated by removing prephenate dehydratase and tuning down the expression level of OPc4CL2. The final strain YRA113-15B produced 208 mg/L rosmarinic acid in a shake-flask culture (a 63-fold improvement over the initial strain), which was the highest rosmarinic acid titer by engineered microbial cells reported to date. This work provides a promising platform for fermentative production of rosmarinic acid and offers a strategy to overcome the intrapathway competition.

Functional insights into two Ocimum kilimandscharicum 4-coumarate-CoA ligases involved in phenylpropanoid biosynthesis.

Plant 4-coumarate-CoA ligase (4CL) catalyzes the ligation of CoA to cinnamic acid and its derivatives. Activated CoA esters are utilized for the biosynthesis of phenolic metabolites and lignin that play essential function in plants. Here, we characterize the diversity of Ocimum kilimandscharicum 4CLs (Ok4CLs). Phylogenetic analysis suggest that Ok4CLs could be grouped into three classes, class I - enzymes mostly involved in lignin biosynthesis, class II - non-structural phenylpropanoid biosynthesis and class III - yet to be characterized for specific role(s). We selected two Ok4CLs namely Ok4CL7 and Ok4CL15 for further characterization. Gene expression analysis suggested that Ok4CL7 is highly expressed in leaf trichomes, whereas Ok4CL15 is abundant in the roots. The recombinant Ok4CL7 and Ok4CL15 had optimal enzyme activities at 40 °C in pH 8 and 7, respectively. Ok4CL7 showed substrate preference towards p-coumaric acid, ferulic acid and caffeic acid. While, Ok4CL15 preferred p-coumaric acid, ferulic acid and sinapic acid. Feruloyl adenylate showed higher number of contacts and lowers binding energy with Ok4CL7 and 15 compared to cinnamoyl adenylate. Based on root-specific expression and preference for sinapic acid, Ok4CL15 might be involved in lignin biosynthesis. Further exploration is needed to unravel the role of diverse Ok4CLs in O. kilimandscharicum.