Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering.

BACKGROUND: Betulinic acid is a pentacyclic lupane-type triterpenoid and a potential antiviral and antitumor drug, but the amount of betulinic acid in plants is low and cannot meet the demand for this compound. Yarrowia lipolytica, as an oleaginous yeast, is a promising microbial cell factory for the production of highly hydrophobic compounds due to the ability of this organism to accumulate large amounts of lipids that can store hydrophobic products and supply sufficient precursors for terpene synthesis. However, engineering for the heterologous production of betulinic acid and related triterpenoids has not developed as systematically as that for the production of other terpenoids, thus the production of betulinic acid in microbes remains unsatisfactory. RESULTS: In this study, we applied a multimodular strategy to systematically improve the biosynthesis of betulinic acid and related triterpenoids in Y. lipolytica by engineering four functional modules, namely, the heterogenous CYP/CPR, MVA, acetyl-CoA generation, and redox cofactor supply modules. First, by screening 25 combinations of cytochrome P450 monooxygenases (CYPs) and NADPH-cytochrome P450 reductases (CPRs), each of which originated from 5 different sources, we selected two optimal betulinic acid-producing strains. Then, ERG1, ERG9, and HMG1 in the MVA module were overexpressed in the two strains, which dramatically increased betulinic acid production and resulted in a strain (YLJCC56) that exhibited the highest betulinic acid yield of 51.87 ± 2.77 mg/L. Then, we engineered the redox cofactor supply module by introducing NADPH- or NADH-generating enzymes and the acetyl-CoA generation module by directly overexpressing acetyl-CoA synthases or reinforcing the ß-oxidation pathway, which further increased the total triterpenoid yield (the sum of the betulin, betulinic acid, betulinic aldehyde yields). Finally, we engineered these modules in combination, and the total triterpenoid yield reached 204.89 ± 11.56 mg/L (composed of 65.44% betulin, 23.71% betulinic acid and 10.85% betulinic aldehyde) in shake flask cultures. CONCLUSIONS: Here, we systematically engineered Y. lipolytica and achieved, to the best of our knowledge, the highest betulinic acid and total triterpenoid yields reported in microbes. Our study provides a suitable reference for studies on heterologous exploitation of P450 enzymes and manipulation of triterpenoid production in Y. lipolytica.

Li2CsB7O10(OH)4: A Deep-Ultraviolet Nonlinear-Optical Mixed-Alkaline Borate Constructed by Unusual Heptaborate Anions.

A new noncentrosymmetric mixed-alkaline borate, Li2CsB7O10(OH)4 (1), was made under solvothermal conditions. This layered boron oxide framework consists of unique bird-shaped [B7O12(OH)4]7- clusters with large 14-ring pores. Its second-harmonic-generation (SHG) signal is 2.5KDP (KH2PO4), with its short ultraviolet (UV) cutoff edge indicating that this crystal is a potential deep-UV transparent nonlinear-optical material.

Galloborates as ultraviolet nonlinear optical crystals: advances and perspectives.

Metal borates are excellent source materials for exploring short-wavelength nonlinear optical (NLO) crystals. Galloborates show rich structural chemistry with various coordination configurations of Ga cation and B-O anionic units and are suitable candidates as ultraviolet NLO crystals. Up to now, the shortest cut-off edge of galloborates was reported to be down to 190 nm in KCs2Ga(B5O10)(OH), while the largest second harmonic generation (SHG) effect of galloborates was reported to be up to 4.6 times that of KH2PO4 (KDP) in Na5Ga[B7O12(OH)]2·2B(OH)3. Herein, we give a detailed summary of the recent progress in NLO inorganic galloborates, where these galloborates are grouped into two types in terms of their compositions: (1) alkali/alkaline earth metal galloborates and (2) alkali/alkaline earth metal galloborate halides. We discuss their structural features, band gaps, and SHG intensities. Finally, we give future perspectives in this field.

[Comparison of the efficiency between in-vitro maturation and in-vitro fertilization after early follicular phase GnRH agonist down-regulation in infertile women with polycystic ovary syndrome].

OBJECTIVE: To compare the outcomes of in-vitro maturation (IVM) and in-vitro fertilization (IVF) after early follicular phase gonadotropin-releasing hormone agonist (GnRH-a) down-regulation in infertile patients with polycystic ovary syndrome (PCOS). METHODS: From July 2010 to December 2012, 72 infertile patients with PCOS undergoing assisted reproductive technology treatment in the Affiliated First Hospital of Wenzhou Medical University were enrolled in this study. The patients were divided into 2 groups, which were patients with early follicular phase down-regulation IVM (36 cases) at IVM group and early follicular phase down-regulation long protocol IVF (36 cases) at IVF group. The laboratory parameters and clinical outcomes were compared between two groups. RESULTS: (1) Lab parameters: a total of 442 oocytes were retrieved in group IVM, and 560 were in group IVF. The rate of mature oocytes of 83.8% (469/560) and high-quality embryos of 70.9% (212/299) at group IVF were significantly higher than that of group IVM[54.1% (239/442) and 50.7% (73/144), retrospectively, P < 0.01]. In group IVM, the average duration of gonadotropin (Gn) was (2.8 ± 1.5) days and the average dosage of Gn was (285 ± 169) U, which were significantly lower than (11.0 ± 1.0) days and (1499 ± 165) U in group IVF (P < 0.01). The mean number of oocytes retrieved 12.8 ± 2.5, fertilization rate of 64.8% (155/239), and implantation rate of 31% (23/74) in group IVM and 15.6 ± 3.1, 65.5% (307/469), 31% (23/74) in group IVF, which did not reach statistical difference (P > 0.05) . (2) Clinical outcomes: the clinical pregnancy rate (17/31, 55%) of IVF group was not significantly higher than that 44% (14/32) at IVM group (P > 0.05). The abortion rate was 1/17 at Group IVF and 1/14 in group IVM, which did not show statistical difference. Women at IVM group has no ovarian hyper-stimulation syndrome (OHSS) cycle, group IVF has 31% (11/36) cycles presented moderate and severe OHSS. CONCLUSIONS: Infertile patients with PCOS undergoing IVM and IVF treatment after early follicular phase GnRH-a down-regulation can get satisfactory laboratory and clinical outcome. In addition to short treatment cycle, IVM can also avoid the occurrence of OHSS completely, but it has a rising trend in the abortion rate. IVF has a high incidence of OHSS, meanwhile, it increases the dosage of gonadotropins.

PRL/PRLR Can Promote Insulin Resistance by Activating the JAK2/STAT5 Signaling Pathway.

Objective: Although prolactin (PRL) is known to affect food intake, weight gain, and insulin resistance, its effects on lipid metabolism and underlying mechanisms remain underinvestigated. This study aimed to investigate the effects of PRL and its receptor (PRLR) on fat metabolism in regulating the JAK2/STAT5 signaling pathway. Methods: SW872 adipocytes were incubated with oleic acid to establish an insulin resistance (IR) model. Western blot was used to detect the expression of PRLR, JAK2, p-JAK2, STAT5, and p-STAT5. Triglyceride (TG) mass was detected by chemical colorimetry methods. Results: Fat droplets in the high-dose and medium-dose PRL groups were significantly higher than in the IR model group. TG mass in the cells was increased significantly compared with the model group. Compared with the control group, the expression of PRLR, p-JAK2, and p-STAT5 were significantly decreased in the IR model group when PRL was intervened for 24 h and 48 h. The expression of PRLR, p-JAK2, and p-STAT5 in the high-dose PRL intervention group increased significantly compared with the model group. The PRLR overexpressing group had significantly increased TG content and PRLR, and JAK2, p-JAK2, STAT5, and p-STAT5 levels compared with the IR model. Conclusion: PRL and PRLR are related to fat metabolism, and the PRL/PRLR signaling pathway can promote insulin resistance by activating the JAK2/STAT5 signaling pathway and increasing the deposition of TGs.

High production of triterpenoids in Yarrowia lipolytica through manipulation of lipid components.

BACKGROUND: Lupeol exhibits novel physiological and pharmacological activities, such as anticancer and immunity-enhancing activities. However, cytotoxicity remains a challenge for triterpenoid overproduction in microbial cell factories. As lipophilic and relatively small molecular compounds, triterpenes are generally secreted into the extracellular space. The effect of increasing triterpene efflux on the synthesis capacity remains unknown. RESULTS: In this study, we developed a strategy to enhance triterpene efflux through manipulation of lipid components in Y. lipolytica by overexpressing the enzyme Δ9-fatty acid desaturase (OLE1) and disturbing phosphatidic acid phosphatase (PAH1) and diacylglycerol kinase (DGK1). By this strategy combined with two-phase fermentation, the highest lupeol production reported to date was achieved, where the titer in the organic phase reached 381.67 mg/L and the total production was 411.72 mg/L in shake flasks, exhibiting a 33.20-fold improvement over the initial strain. Lipid manipulation led to a twofold increase in the unsaturated fatty acid (UFA) content, up to 61-73%, and an exceptionally elongated cell morphology, which might have been caused by enhanced membrane phospholipid biosynthesis flux. Both phenotypes accelerated the export of toxic products to the extracellular space and ultimately stimulated the capacity for triterpenoid synthesis, which was proven by the 5.11-fold higher ratio of extra/intracellular lupeol concentrations, 2.79-fold higher biomass accumulation and 2.56-fold higher lupeol productivity per unit OD in the modified strains. This strategy was also highly efficient for the biosynthesis of other triterpenes and sesquiterpenes, including α-amyrin, ß-amyrin, longifolene, longipinene and longicyclene. CONCLUSIONS: In conclusion, we successfully created a high-yield lupeol-producing strain via lipid manipulation. We demonstrated that the enhancement of lupeol efflux and synthesis capacity was induced by the increased UFA content and elongated cell morphology. Our study provides a novel strategy to promote the biosynthesis of valuable but toxic products in microbial cell factories.

Mono-ADP-ribosylation of histone 3 at arginine-117 promotes proliferation through its interaction with P300.

Relatively little attention has been paid to ADP-ribosylated modifications of histones, especially to mono-ADP-ribosylation. As an increasing number of mono-ADP-ribosyltransferases have been identified in recent studies, the functions of mono-ADP-ribosylated proteins have aroused research interest. In particular, histones are substrates of some mono-ADP-ribosyltransferases and mono-ADP-ribosylated histone have been detected in physiological or pathological processes. In this research, arginine-117 (Arg-117; R-117) of hsitone3(H3) is identified as the a site of mono-ADP-ribosylation in colon carcinoma(the first such site to be identified); this posttranslational modification may promote the proliferation of colon carcinoma cells in vitro and in vivo. Using a point-mutant lentivirus transfection and using an activator of P300 allowed us to observe the mono-ADP-ribosylation at H3R117 and enhancement of the activity of P300 to up-regulate the level of acetylated ß-catenin, which could increase the expression of c-myc and cyclin D1.

Regulation of the RhoA/ROCK/AKT/ß-catenin pathway by arginine-specific ADP-ribosytransferases 1 promotes migration and epithelial-mesenchymal transition in colon carcinoma.

Arginine-specific ADP-ribosytransferases 1 (ART1) is able to modify the arginine of specific proteins by mono-ADP-ribosylation. We previously reported that the expression of ART1 in human colon adenocarcinoma tissues was higher than in adjacent tissues. Herein, we primarily revealed that ART1 could regulate the epithelial-mesenchymal transition (EMT) and, therefore, the development of colon carcinoma. In CT26 cells, which overexpressed ART1 by lentiviral transfection, the following were promoted: alterations of spindle-like non-polarization, expression of EMT inducers and mesenchymal markers, migration, invasion and adhesion. However, epithelial marker expression was decreased. Correspondingly, knockdown of ART1 in CT26 cells had the opposite effects. The effect of ART1 on EMT and carcinoma metastasis was also verified in a liver metastasis model of BALB/c mice. To further explore the molecular mechanism of ART1 in EMT, CT26 cells were treated with several specific inhibitors and gene silencing. Our data suggest that ART1 could regulate EMT by regulating the RhoA/ROCK1/AKT/ß-catenin pathway and its downstream factors (snail1, vimentin, N-cadherin and E-cadherin) and that it therefore plays an important role in the progression of colon carcinoma.