Development of the Static and Dynamic Gene Expression Regulation Toolkit in Pseudomonas chlororaphis.

The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism Pseudomonas chlororaphis is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes lacZ encoding ß-galactosidase from Escherichia coli K12 and phzO encoding PCA monooxygenase from P. chlororaphis GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed PMOK_RS0128085UTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity PMOK_RS0116635UTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for P. chlororaphis, providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.

Comparative metabolomics and transcriptomics analyses provide insights into the high-yield mechanism of phenazines biosynthesis in Pseudomonas chlororaphis GP72.

AIMS: Phenazines, such as phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA), 2-hydroxyphenazine (2-OH-PHZ), are a class of secondary metabolites secreted by plant-beneficial Pseudomonas. Ps. chlororaphis GP72 utilizes glycerol to synthesize PCA, 2-OH-PCA and 2-OH-PHZ, exhibiting broad-spectrum antifungal activity. Previous studies showed that the addition of dithiothreitol (DTT) could increase the phenazines production in Ps. chlororaphis GP72AN. However, the mechanism of high yield of phenazine by adding DTT is still unclear. METHODS AND RESULTS: In this study, untargeted and targeted metabolomic analysis were adopted to determine the content of metabolites. The results showed that the addition of DTT to GP72AN affected the content of metabolites of central carbon metabolism, shikimate pathway and phenazine competitive pathway. Transcriptome analysis was conducted to investigate the changed cellular process, and the result indicated that the addition of DTT affected the expression of genes involved in phenazine biosynthetic cluster and genes involved in phenazine competitive pathway, driving more carbon flux into phenazine biosynthetic pathway. Furthermore, genes involved in antioxidative stress, phosphate transport system and mexGHI-opmD efflux pump were also affected by adding DTT. CONCLUSION: This study demonstrated that the addition of DTT altered the expression of genes related to phenazine biosynthesis, resulting in the change of metabolites involved in central carbon metabolism, shikimate pathway and phenazine competitive pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: This work expands the understanding of high yield of phenazine by the addition of DTT and provides several targets for increasing phenazine production.

Acmoxanthones A-E, New Lavandulated Xanthones from Hypericum acmosepalum N. Robson.

Acmoxanthones A-E (1-5), five new lavandulylated xanthones, were isolated from the aerial parts of Hypericum acmosepalum, together with four known xanthones. Their structures with absolute configurations were elucidated on the basis of analysis of MS, NMR and chiroptical properties. A bioassay against high glucose-induced damage on human umbilical vein endothelial cells (HUVECs) showed ananixanthone (6) and osajaxanthone (7) had potential antioxidative damage activity with EC50 values of 10.5 µg/mL and 7.6 µg/mL, respectively, while 3-hydroxy-2,4-dimethoxyxanthone (8) exhibited cytotoxic effect on the damaged cells with IC50 values of 7.1 µg/mL.

Structurally diverse polyprenylated acylphloroglucinols from Hypericum uralum Buch.-Ham. ex D. Don.

Uralins A - D, four undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs) featuring an unprecedented fused hexacyclic architecture, a unique monocyclic tetra-seco-tetranor-b-PPAP, an oxidative b-PPAP and a rare norspiroindane-type m-PPAP, respectively, were isolated from the aerial parts of Hypericum uralum, along with ten known PPAPs. Their structures and absolute configurations were elucidated by extensive spectroscopic techniques (MS, NMR, [α]D, CD), conceivable biogenetic pathways and time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations. Biological assays showed three b-PPAPs had moderate antioxidative damage activities, while spiroindanes exhibited moderate cytotoxic effects.

Characterization and Engineering of Pseudomonas chlororaphis LX24 with High Production of 2-Hydroxyphenazine.

The take-all disease of wheat is one of the most serious diseases in the field of food security in the world. There is no effective biological pesticide to prevent the take-all disease of wheat. 2-Hydroxyphenazine (2-OH-PHZ) was reported to possess a better inhibitory effect on the take-all disease of wheat than phenazine-1-carboxylic acid, which was registered as "Shenqinmycin" in China in 2011. The aim of this study was to construct a 2-OH-PHZ high-producing strain by strain screening, genome sequencing, genetic engineering, and fermentation optimization. First, the metabolites of the previously screened new phenazine-producing Pseudomonas sp. strain were identified, and the taxonomic status of the new Pseudomonas sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new Pseudomonas sp. strain was named Pseudomonas chlororaphis subsp. aurantiaca LX24, which is a new subspecies of P. chlororaphis that can synthesize 2-OH-PHZ. Next, the draft genome of strain LX24 was determined, and clusters of orthologous group (COG) analysis, KEGG analysis, and gene ontology (GO) analysis of strain LX24 were performed. Furthermore, the production of 2-OH-PHZ increased to 351.7 from 158.6 mg/L by deletion of the phenazine synthesis negative regulatory genes rpeA and rsmE in strain LX24. Finally, the 2-OH-PHZ production of strain LX24 reached 677.1 mg/L after fermentation optimization, which is the highest production through microbial fermentation reported to date. This work provides a reference for the efficient production of other pesticides and antibiotics.

Engineering of glycerol utilization in Pseudomonas chlororaphis GP72 for enhancing phenazine-1-carboxylic acid production.

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.

Two new noroleanane-type triterpenoid saponins from the stems of Stauntonia chinensis.

Two new noroleanane-type triterpenoid saponins, 3ß,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 24-O-ß-L-fucopyranosyl-(1→2)-[ß-D-xylopyranosyl-(1→3)]-ß-D-glucopyranoside (1) and 3ß,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 24-O-ß-D-glucopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-ß-D-glucopyranoside (2) were isolated from the stems of Stauntonia chinensis DC., together with three known compounds, brachyantheraoside B2 (3), eupteleasaponin Ⅷ (4) and fargoside B (5). Their structures were elucidated by spectroscopic and chemical methods. The cytotoxic activities of compounds 1 and 2 were evaluated against five human tumor cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780). Compounds 1 and 2 showed moderate cytotoxic activities toward the tested cell lines with IC50 values ranging from 12.71 to 32.04 µM.

Cytotoxic triterpenoid saponins from the defatted seeds of Camellia oleifera Abel.

Five new oleanane-type triterpenoid saponins, oleiferasaponins D1-D5 (1-5), were isolated from the defatted seeds of Camellia oleifera Abel. Their structures were elucidated by spectroscopic and chemical methods. The cytotoxic activities of compounds 1-5 were evaluated against five human tumor cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780). Compounds 1-2 exhibited cytotoxic activity against five human cancer cell lines, with IC50 values ranging from 3.31 to 10.23 µM. Compounds 3-5 showed moderate cytotoxic activities toward the tested cell lines.

DNA hypermethylation of the vimentin gene inversely correlates with vimentin expression in intestinal- and diffuse-type gastric cancer.

The vimentin gene is a hallmark of epithelial-to-mesenchymal transition and has been observed to be overexpressed in various types of tumor cell line and tissue. Previous studies have reported correlations between vimentin DNA methylation levels and subsequent vimentin expression levels in solid tumors, including breast and colorectal cancer; however, to the best of our knowledge, such a correlation has not been reported for gastric cancer (GC) using Lauren classification. Therefore, the present study aimed to quantify DNA methylation levels of the vimentin gene using quantitative (q) methylation-specific polymerase chain reaction (PCR) in intestinal-type GC cell lines (MKN-28, AGS and MKN-1), diffuse-type GC cell lines (SGC-7901, SNU-5 and KATO III), the GES-1 immortalized human non-neoplastic gastric epithelial cell line, as well as in tumor and paratumor normal tissue samples. Furthermore, the present study analyzed the messenger RNA expression of the vimentin gene in these cell lines and tissues by reverse transcription-qPCR. A comparison of the clinicopathological features was conducted between patients, grouped according to the Lauren classification. The present study identified that the vimentin promoter region was hypermethylated in all GC cell lines and tumor tissue samples when compared with immortalized normal gastric epithelial cells and paratumor normal tissues. In addition, vimentin promoter methylation levels were observed to be higher in intestinal-type cell lines when compared with those of diffuse-type lines and tissues. Correspondingly, vimentin expression levels were lower in intestinal-type gastric cell lines compared with those of diffuse-type cell lines and tissues, and were lowest in the non-neoplastic gastric cell line and paratumor normal tissues. Patients with diffuse-type GC were on average younger (P=0.023), and exhibited higher tumor (P=0.020), node (P=0.032) and TNM classification of malignant tumor stage (P=0.039) than those with intestinal-type GC. Following treatment of AGS cells (which demonstrated the highest methylation level of the vimentin gene) with 5-aza-2'-deoxycytidine, vimentin expression was restored significantly. Thus, the present study revealed that vimentin promoter methylation levels are inversely correlated with vimentin expression levels in GC (according to Lauren classification). High levels of methylation in the vimentin gene promoter region may be involved in carcinogenesis and the development of GC, and may provide a novel molecular classification for GC.