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Two novel Bacillus glycoside hydrolase family 53 (GH53) endo-1,4-β-galactanases (Bs936 and Bs4828) were identified. The recombinant Bs936 and Bs4828 displayed maximal activities at pH 5.5/55 °C and pH 6.5/65 °C, respectively. Stability analyses revealed that the enzymes were stable between pH 4.5-10, retaining over 80% activities after 12 h incubation at 25 °C. Moreover, Bs936 maintained about 75% activity after being treated at 45 °C for 2 h, while Bs4828 kept full activity after 4 h of incubation at 50 °C. Importantly, Bs936 and Bs4828 exhibited good activity towards β-1,4-galactan, showing specific activities of 1859.46- and 3110.79 U/mg towards potato galactan, respectively. The corresponding K

GH42 enzymes are potential candidates for bifunctional β-galactosidase/α-L-arabinopyranosidase. A novel GH42 enzyme (BaBgal42A) from Bacillus was identified, the recombinant BaBgal42A hydrolyzed not only β-D-galactopyranosidic bonds in pNP-β-D-galactopyranoside, oNP-β-D-galactopyranoside, lactose, galactan, and arabinan but also α-L-arabinopyranosidic linkages in pNP-α-L-arabinopyranoside, wheat arabinoxylan and galactan. The Km values of BaBgal42A for pNP-β-D-galactopyranoside and pNP-α-L-arabinopyranoside were 2.76 and 16.23 mM, respectively. Investigation of cooperative activities of BaBgal42A with cognate enzymes revealed that BaBgal42A showed obvious synergy with an endo-β-1,4-galactanase (BaGal53A) in the decomposition of galactan, supplementing BaBgal42A resulted in a 0.56-fold increase in the release of reducing sugars; BaBgal42A also exhibited a little synergy with its cognate endoxylanase (BaXynA)/α-L-arabinofuranosidase (BaAraA) in hydrolyzing wheat arabinoxylan/arabinan, addition of BaBgal42A released 12.7%/7.8% more reducing sugars than that produced by BaXynA/BaAraA alone. Moreover, BaBgal42A is a cold-adapted enzyme, exhibiting 28–46% of the maximal activity at the range of 5–20 °C and its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. This study not only expands the diversity within GH42 family, but also provides new insights into the role of microbial GH42 enzymes, which would contribute to its potential application in polysaccharides degradation and milk lactose hydrolysis.

Andrographis paniculata (Burm. f.) Nees is one of the most important economic crops for its various medicinal properties, which has been widely used as medicinal herb in several folk medicine systems all over the world. A. paniculata suffers from continuous cropping problem in cultivation; however, effects of continuous cropping on A. paniculata and the underlying mechanism at molecular level are rarely understood. In this study, Illumina paired-end RNA-seq was performed for de novo transcriptome construction of A. paniculata leaf with or without 2-years continuous cropping, to identify gene expression in response to continuous cropping stress. A total of 43,683 Unigenes were obtained by de novo assembly of 231.53 million paired-end clean reads. RNA-seq based gene expression profiling showed a total of 6193 Unigenes was significantly up-/down-regulated after continuous cropping. Integrating the GO and KEGG enrichment analysis of differentially expressed genes (DEGs) revealed that A. paniculata could alter enzyme genes expression to adjust the complicated metabolic pathways to tolerate the continuous cropping stress. Specially, accorded with the downtrend of active component contents in A. paniculata, majority of genes involved in the terpenoids biosynthesis, phenylpropanoid biosynthesis and flavonoids biosynthesis pathways were down-regulated, indicating that continuous cropping led to a declined synthesis of active ingredients through repressing the expression levels of genes involved in these metabolites’ biosynthesis pathways. The transcriptome profiling will deepen the understanding of A. paniculata under continuous cropping stress at transcriptional level, and provide useful genomic resource for further interpretation on the regulation mechanism.

Chrysanthemum indicum L. is a type of herb that is widely used in China, Korea, and Japan. It has been used as an ingredient in traditional medicines, tea, and functional food because of its various anti-inflammatory and anti-oxidant bioactivities. Such bioactivities have been associated with flavonoids such as apigenin, luteolin and linarin in C. indicum. However, the biosynthesis pathway has not been investigated. In this study, using transcriptomic analysis and targeted metabolic profiling from five different tissues, we characterize the levels of flavonoids and mine the corresponding genes involved in flavonoid biosynthesis. Transcriptomic analysis revealed that 103 unigenes are involved in flavonoid-related biosynthesis pathways. Flavone synthase (FNS) is the key enzyme responsible for flavone synthesis and provides precursors for acacetin and linarin biosynthesis. One putative FNS Ⅱ gene, with the highest Reads Per Kilobase per Million mapped reads (RPKM) in flower and flower bud was cloned. Quantitative real-time polymerase chain reaction (RT-qPCR) revealed that CiFNSⅡ exhibited a similar expression pattern to that in the transcriptome in terms of RPKM. In addition, a targeted metabolic profiling of three flavanones (naringenin, eriodictyol, and liquiritigenin), three flavones (apigenin, luteolin, and 7,4′-dihydroxyflavone), and two flavone derivatives (linarin and acacetin) was performed to characterize the distribution of these flavonoids in different tissues of C. indicum. The recombinant FNSⅡ protein expressed in yeast was able to catalyze the conversion of three flavanones into the respective flavones. Based on the transcriptome analysis, metabolic profiling, and activity assays, a linarin biosynthesis pathway is proposed. Our study provides insight into the potential application of molecular breeding and metabolic engineering for improving the quality of cultivated C. indicum.

The anthraquinones endocrocin and emodin are synthesized by a special class of type I NR-PKSs and a discrete MβL-TE. In this work, we first reconstituted a biosynthetic pathway of endocrocin and emodin in S. cerevisiae by combining enzymes from different sources. We functionally characterized a TE-less NR-PKS (SlACAS) and a MβL-TE (SlTE) from S. lycopersici as well as four orthologous MβL-TEs. SlACAS was coexpressed with different MβL-TEs in S. cerevisiae. SlACAS generated the highest amount of endocrocin when coupled with HyTE, the yield was 115.6% higher than that with the native SlTE. To accumulate more emodin, seven decarboxylases with high homology to HyDC were identified and introduced into the biosynthetic pathway. Among these orthologs, AfDC exhibited the highest catalytic activity and the conversion rate reached 98.6%. A double-point mutant acetyl-CoA carboxylase, ACC1S659A, S1157A, was further introduced to increase the production of malonyl-CoA as a precursor of these anthraquinones. The production of endocrocin (233.6 ± 20.3 mg/L) and emodin (253.2 ± 21.7 mg/L) then dramatically increased. We also optimized the carbon source in the medium and conducted fed-batch fermentation with the engineered strains. The titers of endocrocin and emodin obtained were 661.2 ± 50.5 mg/L and 528.4 ± 62.7 mg/L, respectively, which are higher than previously reported. In this work, by screening a small library of orthologous biosynthetic bricks, an efficient biosynthetic pathway of endocrocin and emodin was first created in S. cerevisiae. This study provides a novel metabolic engineering approach for optimization of the production of desired molecules.

Iridoid compounds have been reported to be accumulated in the dried fruits of Gardenia jasminoides (Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl- d -erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.

Trihelix (TH) transcription factors (TFs) actively function in regulating growth and development of plant. Moreover, members of this gene family regulate environment-responsive secondary metabolism. Here, members of trihelix family were explored based on the full-length transcriptome of Pogostemon cablin (Blanco) Benth. (patchouli), an extensively used medicinal plant. Among them, 16 PatTHs exhibited complete opening reading frame (ORF) were successfully cloned, and were classified according to their structural properties into four subfamilies (GT-1, GT-2, SH4, and SIP1). The expression patterns of PatTHs varied in various P. cablin tissues, and most of the PatTHs were induced by methyl jasmonate (MeJA). On exposure to abiotic stresses, including salt, drought, and cold condition, each PatTH responded to at least one of the stresses. In addition, the promoter of patchouli HMGR gene was bound and repressed by PatGT-1, a homologous protein of Arabidopsis transcription factor GT-1. According to subcellular localization results, PatGT-1 is a nuclear-localized protein. Once PatGT-1 was transiently overexpressed, it significantly lowered the production of patchoulol via repressing genes in patchoulol biosynthetic pathway. In this study, PatTHs and their putative functions were identified from P. cablin, and PatGT-1 was revealed as a negative regulator in patchoulol synthesis. Our findings improve the understanding of trihelix family in patchouli and contribute to the future study on this family.

Patchoulol, the major active ingredient in Pogostemon cablin (Blanco) Benth., has considerable clinical and industrial value. A number of genes that participate in the patchoulol biosynthesis pathway have been identified; however, little is known about the transcription factors involved in regulating patchoulol synthesis. In this study, PatSWC4, a homologous protein of Arabidopsis SWC4 belonging to the MYB (v-myb avian myeloblastosis viral oncogene homolog)-related transcription factor family, was identified and characterized. Subcellular localization showed that PatSWC4 protein localized in the nucleus. The highest expression level of PatSWC4 gene was found in old leaves of P. cablin and it was significantly induced by methyl jasmonate (MeJA). Additionally, yeast one-hybrid (Y1H) and dual-luciferase (dual-LUC) assays revealed that PatSWC4 could bind to the promoter of PatPTS gene to increase its transcriptional activity. Yeast two-hybrid (Y2H) assays also confirmed the interaction between PatSWC4 and PatJAZ4 proteins, indicating that PatSWC4 might function in the JA response network. Moreover, transient overexpression of PatSWC4 gene in P. cablin leaves markedly increased the production of patchoulol, and qRT-PCR analysis further revealed that genes in the patchoulol biosynthesis pathway were significantly upregulated at the transcriptional level. Ultimately, we proposed a work model depicting how JA signaling regulates PatPTS via the interaction between PatSWC4 and PatJAZ4, thereby regulating the biosynthesis of patchoulol. Our findings not only help to elucidate the regulatory mechanism governing JA-induced patchoulol biosynthesis, but also lay a foundation for future studies on improving patchoulol production through genetic and metabolic engineering.

STAT3 signaling pathway is an important target for human cancer therapy. Thus, the identification of small-molecules that target STAT3 signaling will be of great interests in the development of anticancer agents. The aim of this study was to identify novel inhibitors of STAT3 pathway from the roots of Zanthoxylum nitidum (Roxb.) DC. The bioassay-guided fractionation of MeOH extract of Z. nitidum using a STAT3-responsive gene reporter assay led to the isolation of angoline (1) as a potent and selective inhibitor of the STAT3 signaling pathway (IC50=11.56 μM). Angoline inhibited STAT3 phosphorylation and its target gene expression and consequently induced growth inhibition of human cancer cells with constitutively activated STAT3 (IC50=3.14-4.72 μM). This work provided a novel lead for the development of anti-cancer agents targeting the STAT3 signaling pathway.

Exogenous hormone application is an effective and feasible method to regulate production of economic plant and synthesis of high-value natural medical products. Jasmonate and ethylene are two crucial plant hormones regulate growth and development, secondary metabolism, and stress responses in Pogostemon cablin. However, the regulatory mechanism of these phytohormones in this important medicinal plant has not been elucidated to date. In the present study, alterations of secondary metabolites and photosynthesis of P. cablin leaves treated with exogenous methyl jasmonate (MeJA), ethrel (ETH), and simultaneously with both MeJA and ETH, respectively, were explored. To uncover the potential protein functional network involved in the hormone-induction mechanism, we performed a proteomic analysis of differently treated leaves using isobaric tags for relative and absolute quantification (iTRAQ). In P. cablin leaves before and after three different treatments, 254, 229 and 400 differentially expressed proteins (DEPs) involving a wide range of physiological processes were clearly identified respectively. The quantitative data were further validated by parallel reaction monitoring (PRM) and qRT-PCR. Functional enrichment analysis indicated photosynthesis-related proteins showed a significant increase expression in P. cablin leaves after MeJA treatment, while proteins involved in secondary metabolite biosynthesis and thiamine metabolism were significantly down-regulated. Exogenous ETH mainly repressed the expression of proteins related to carbohydrate and energy metabolism in P. cablin leaves while markedly raised protein abundance in photosynthesis and protein processing in the endoplasmic reticulum. Regarding P. cablin leaves treated with both MeJA and ETH, expression of proteins related to carbohydrate and energy metabolism, secondary metabolite biosynthesis, tyrosine metabolism, thiamine metabolism and lipid metabolism were significantly down-regulated, while that of proteins involved in photosynthesis, phenylpropanoid biosynthesis and nitrogen metabolism were considerably up-regulated. This is the first study using iTRAQ-based proteomics approach to comprehensively elucidate exogenous hormone response of P. cablin, providing important pathways/proteins involved in secondary metabolism regulation, which will contribute to potential genetic improvement of medical constituents.