Establishment of a selectable marker recycling system for iterative gene editing in Fusarium fujikuroi.

Gibberellic acid (GA3) is a vital plant growth hormone widely used in agriculture. Currently, GA3 production relies on liquid fermentation by the filamentous fungus Fusarium fujikuroi. However, the lack of an effective selection marker recycling system hampers the application of metabolic engineering technology in F. fujikuroi, as multiple-gene editing and positive-strain screening still rely on a limited number of antibiotics. In this study, we developed a strategy using pyr4-blaster and CRISPR/Cas9 tools for recycling orotidine-5'-phosphate decarboxylase (Pyr4) selection markers. We demonstrated the effectiveness of this method for iterative gene integration and large gene-cluster deletion. We also successfully improved GA3 titers by overexpressing geranylgeranyl pyrophosphate synthase and truncated 3-hydroxy-3-methyl glutaryl coenzyme A reductase, which rewired the GA3 biosynthesis pathway. These results highlight the efficiency of our established system in recycling selection markers during iterative gene editing events. Moreover, the selection marker recycling system lays the foundation for further research on metabolic engineering for GA3 industrial production.

Improving the productivity of gibberellic acid by combining small-molecule compounds-based targeting technology and transcriptomics analysis in Fusarium fujikuroi.

Gibberellic acid (GA3), produced industrially by Fusarium fujikuroi, stands as a crucial plant growth regulator extensively employed in the agriculture filed while limited understanding of the global metabolic network hinders researchers from conducting rapid targeted modifications. In this study, a small-molecule compounds-based targeting technology was developed to increase GA3 production. Firstly, various small molecules were used to target key nodes of different pathways and the result displayed that supplement of terbinafine improved significantly GA3 accumulation, which reached to 1.08 g/L. Subsequently, lipid and squalene biosynthesis pathway were identified as the key pathways influencing GA3 biosynthesis by transcriptomic analysis. Thus, the strategies including in vivo metabolic engineering modification and in vitro supplementation of lipid substrates were adopted, both contributed to an enhanced GA3 yield. Finally, the engineered strain demonstrated the ability to achieve a GA3 yield of 3.24 g/L in 5 L bioreactor when utilizing WCO as carbon source and feed.

Improving Gibberellin GA3 Production with the Construction of a Genome-Scale Metabolic Model of Fusarium fujikuroi.

Liquid fermentation is the primary method for GA3 production usingFusarium fujikuroi. However, production capacity is limited due to unknown metabolic pathways. To address this, we constructed a genome-scale metabolic model (iCY1235) with 1753 reactions, 1979 metabolites, and 1235 genes to understand the GA3 regulation mechanisms. The model was validated by analyzing growth rates under different glucose uptake rates and identifying essential genes. We used the model to optimize fermentation conditions, including carbon sources and dissolved oxygen. Through the OptForce algorithm, we identified 20 reactions as targets. Overexpressing FFUJ_02053 and FFUJ_14337 resulted in a 37.5 and 75% increase in GA3 titers, respectively. These targets enhance carbon flux toward GA3 production. Our model holds promise for guiding the metabolic engineering of F. fujikuroi to achieve targeted overproduction. In summary, our study utilizes the iCY1235 model to understand GA3 regulation, optimize fermentation conditions, and identify specific targets for enhancing GA3 production through metabolic engineering.

Recent Development of Advanced Biotechnology in the Oleaginous Fungi for Arachidonic Acid Production.

Arachidonic acid is an essential ω-6 polyunsaturated fatty acid, which plays a significant role in cardiovascular health and neurological development, leading to its wide use in the food and pharmaceutical industries. Traditionally, ARA is obtained from deep-sea fish oil. However, this source is limited by season and is depleting the already threatened global fish stocks. With the rapid development of synthetic biology in recent years, oleaginous fungi have gradually attracted increasing attention as promising microbial sources for large-scale ARA production. Numerous advanced technologies including metabolic engineering, dynamic regulation of fermentation conditions, and multiomics analysis were successfully adapted to increase ARA synthesis. This review summarizes recent advances in the bioengineering of oleaginous fungi for ARA production. Finally, perspectives for future engineering approaches are proposed to further improve the titer yield and productivity of ARA.

RETRACTED ARTICLE: Mutagenesis combined with fermentation optimization to enhance gibberellic acid GA3 yield in Fusarium fujikuroi.

Gibberellic acid (GA3) is a plant growth hormone that plays an important role in the production of crops, fruits, and vegetables with a wide market share. Due to intrinsic advantages, liquid fermentation of Fusarium fujikuroi has become the sole method for industrial GA3 production, but the broader application of GA3 is hindered by low titer. In this study, we combined atmospheric and room-temperature plasma (ARTP) with ketoconazole-based screening to obtain the mutant strain 3-6-1 with high yield of GA3. Subsequently, the medium composition and fermentation parameters were systematically optimized to increase the titer of GA3, resulting in a 2.5-fold increase compared with the titer obtained under the initial conditions. Finally, considering that the strain is prone to substrate inhibition and glucose repression, a new strategy of fed-batch fermentation was adopted to increase the titer of GA3 to 575.13 mg/L, which was 13.86% higher than the control. The strategy of random mutagenesis combined with selection and fermentation optimization developed in this study provides a basis for subsequent research on the industrial production of GA3.

YALIcloneNHEJ: An Efficient Modular Cloning Toolkit for NHEJ Integration of Multigene Pathway and Terpenoid Production in Yarrowia lipolytica.

Non-homologous end-joining (NHEJ)-mediated random integration in Yarrowia lipolytica has been demonstrated to be an effective strategy for screening hyperproducer strains. However, there was no multigene assembly method applied for NHEJ integration, which made it challenging to construct and integrate metabolic pathways. In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Y. lipolytica. By optimizing key factors, including the amounts of ligase and the reaction cycles, the assembly efficiency of 4, 7, and 10 fragments reached up to 90, 75, and 50%, respectively. This YALIcloneNHEJ system was subsequently applied for the overproduction of the sesquiterpene (-)-α-bisabolol by constructing a biosynthesis route and enhancing the flux in the mevalonate pathway. The resulting strain produced 4.4 g/L (-)-α-bisabolol, the highest titer reported in yeast to date. Our study expands the toolbox of metabolic engineering and is expected to enable a highly efficient production of various terpenoids.

LINK-A lncRNA is upregulated in osteosarcoma and regulates migration, invasion and stemness of osteosarcoma cells.

The present study aimed to investigate the involvement of long intergenic non-coding RNA for kinase activation (LINK-A) long non-coding RNA (lncRNA) in osteosarcoma. Plasma levels of LINK-A lncRNA and transforming growth factor ß1 (TGF-ß1) were measured by reverse transcription-quantitative polymerase chain reaction and ELISA, respectively. Correlation between LINK-A lncRNA and TGF-ß1 was analyzed by Pearson correlation coefficient. LINK-A lncRNA and TGF-ß1 were upregulated in patients with osteosarcoma compared with healthy controls. LINK-A lncRNA and TGF-ß1 were positively correlated in the two groups. LINK-A lncRNA short hairpin RNAs (shRNAs) were transfected into osteosarcoma cell lines and Transwell migration assay, Matrigel invasion assay and flow cytometry were used to evaluate cell migration, invasion and stemness, respectively. Effects of LINK-A lncRNA silencing and overexpression on TGF-ß1 expression were analyzed by western blotting. LINK-A lncRNA shRNA silencing inhibited, whereas TGF-ß1 treatment promoted cell migration, invasion and stemness. LINK-A lncRNA silencing inhibited TGF-ß1 expression, whereas TGF-ß1 treatment had no effects on LINK-A lncRNA expression. TGF-ß1 reduced the inhibitory effects of LINK-A lncRNA knockdown on cancer cell migration, invasion and stemness. These data indicated that LINK-A lncRNA is upregulated in osteosarcoma and may regulate migration, invasion and stemness of osteosarcoma cells through TGF-ß1.

MiR-671-5p plays a promising role in restraining osteosarcoma cell characteristics through targeting TUFT1.

The aim of our study was to explore the roles of miR-671-5p in mediating biological processes of osteosarcoma (OS) cells and clinical implications. On the basis of the OS samples acquired from the GEO database, the expression difference and overall survival analyses of miR-671-5p and TUFT1 were determined. The expression of MiR-671-5p was verified using OS cell lines. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound-healing, and Transwell assays were respectively carried out to probe whether miR-671-5p regulated OS cell vitality, migration, and invasion. The expression of miR-671-5p was downregulated in OS tissues and cell lines. High expression of MiR-671-5p blocked OS cell growth, migration, and invasion. TUFT1 was predicted and validated as the target of miR-671-5p in OS cells using in silico analysis and luciferase reporter assays. Forced expression of TUFT1 reversed the suppressive influence of miR-671-5p on cell viability, migration, and invasion of OS cells. Moreover, the low expression of miR-671-5p and the high expression of TUFT1 led to poor prognosis. Taken together, targeting miR-671-5p/TUFT1 may be a promising strategy for treating OS.

Zinc promotes cell apoptosis via activating the Wnt-3a/ß-catenin signaling pathway in osteosarcoma.

BACKGROUND: The zinc content in the blood and tumor tissues of patients with osteosarcoma and the underlying regulation and molecular mechanism of zinc have not been reported. METHODS AND RESULTS: This study showed that the zinc content in the blood and tumor tissues of patients with osteosarcoma significantly reduced. CCK-8 and Transwell chamber assays revealed that zinc treatment significantly inhibited the proliferation and invasion abilities of osteosarcoma cells. Western blot analysis indicated that the expression levels of caspase-3 and caspase-9 were significantly increased, suggesting that zinc inhibited the growth and promoted the apoptosis of osteosarcoma cells. In addition, the expression levels of Wnt-3a and ß-catenin, the marker proteins of the Wnt/ß-catenin signaling pathways, were significantly increased in osteosarcoma cells after zinc intervention, which demonstrated that the pathway was clearly activated. However, the effect of zinc on the apoptosis, proliferation, and invasion abilities of osteosarcoma cells was reversed when the Wnt/ß-catenin signaling pathways was inhibited by XAV939 (Wnt antagonist) treatment. CONCLUSIONS: This study is the first to report the changes in zinc levels in the blood and tumor tissues of patients with osteosarcoma and to preliminarily verify that zinc inhibits the proliferation and invasion and promote the apoptosis of osteosarcoma cells by inducing the Wnt/ß-catenin signaling pathway, which ultimately inhibit cancer growth.

MiRNA-125a-5p attenuates blood-spinal cord barrier permeability under hypoxia in vitro.

Disruption of the blood-spinal cord barrier (BSCB) results in secondary injury and apoptosis of neurons, leading to permanent neurological dysfunction after spinal cord injury. In this study, we evaluate the role of miRNA-125a-5p in the BSCB under hypoxia. The miRNA-125a-5p mimics group showed lower horseradish peroxidase (HRP) permeability and endothelial cell death rates compared with the transfection control group. By contrast, the miRNA-125a-5p inhibitor group demonstrated higher HRP permeability and endothelial cell death rates than the transfection control group. The expressions of ZO-1, occludin, VE-cadherin and their mRNA significantly increased in miRNA-125a-5p-overexpressing cells. By contrast, a remarkable reduction in ZO-1, occludin, and VE-cadherin expression and their mRNA were observed in miRNA-125a-5p-inhibited cells. MiRNA-125a-5p appears to reduce the permeability of the BSCB by up regulating the expression of ZO-1, occludin, and VE-cadherin and their mRNA, and against hypoxia-induced apoptosis of spinal cord microvascular endothelial cells. Taken together, our results clearly indicate that miRNA-125a-5p plays an important role in protecting the functions of the BSCB under hypoxia.

MiR-320a was highly expressed in postmenopausal osteoporosis and acts as a negative regulator in MC3T3E1 cells by reducing MAP9 and inhibiting PI3K/AKT signaling pathway.

BACKGROUND: Postmenopausal osteoporosis (PMO), as a frequent disease in postmenopausal women, is mainly caused by the lack of estrogen. MiR-320a has been found to abate osteoblast function and induce oxidative stress before osteoporosis. However, studies on the downstream target gene and related signaling pathway of miR-320a in PMO are still obscure. This study aims to deal with these problems. METHODS: The expression levels of miR-320a and microtubule-associated protein 9 (MAP9) in patients with osteoporosis were analyzed on the basis of the GEO database. The cells viability was determined by methylthiazolyl tetrazolium bromide (MTT). Flow cytometry and western blot were used to detect the cells apoptosis and the expression of apoptosis-related proteins, respectively. The cells differentiation-related proteins were detected by qRT-PCR and western blot. The interaction between miR-320a and MAP9 was predicted by biological software and verified by dual luciferase reporter assay and rescue assay. The expression levels of PI3K, p-PI3K, AKT and p-AKT in MC3T3-E1 cells were assessed by western blot. RESULTS: We observed that miR-320a was over-expressed in PMO patients and exhibited inhibitory effects on MC3T3-E1 cells activity and differentiation, as well as promoting effects on MC3T3-E1 cells apoptosis. MAP9 was verified as a target gene of miR-320a and was negatively regulated by miR-320a. Based on the GEO database, MAP9 was found to be lower expressed in PMO patients. Rescue assay demonstrated that down-regulation of MAP9 could alleviate the promoting effects of miR-320a inhibitor on MC3T3-E1 cells activity and differentiation and the inhibitory effects of miR-320a inhibitor on MC3T3-E1 cells apoptosis. Mechanically, miR-320a/MAP9 possibly took part in MC3T3-E1 cells viability, differentiation and apoptosis via mediating PI3K/AKT signaling pathway. CONCLUSIONS: Our outcomes demonstrated that miR-320a promoted MC3T3-E1 cells apoptosis, suppressed MC3T3-E1 cells viability and differentiation through targeting MAP9 and modulating PI3K/AKT signaling pathway, which provided theoretical support for miR-320a/MAP9 as promising targets for the treatment and prevention of PMO.

Sitagliptin ameliorates advanced glycation end-product (AGE)-induced degradation of extracellular matrix in human primary chondrocytes.

Accumulation of advanced glycation end-products (AGEs) increases inflammation and triggers processes involved in the pathogenesis of osteoarthritis (OA). As a major debilitating age-related disease, it is imperative that novel therapies for OA be sought. In the present study, we investigated the effects of the selective dipeptidyl peptidase IV (DPP-4) inhibitor sitagliptin in human primary chondrocytes exposed to insult by AGEs to elucidate the potential role of sitagliptin in the treatment of OA. Our findings show that inhibition of DPP-4 by sitagliptin could reduce oxidative stress, increase cell viability and prevent degradation of type II collagen and aggrecan by matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) induced by AGEs in human primary chondrocytes. Mechanistically, we found that sitagliptin inhibited AGEs-induced nuclear translocation of p65 protein and drastically decreased the luciferase activity of NF-κB. These findings indicate that sitagliptin may have potential as a novel therapeutic option for the treatment and prevention of OA.

Strontium inhibits titanium particle-induced osteoclast activation and chronic inflammation via suppression of NF-κB pathway.

Wear-particle-induced chronic inflammation and osteoclastogenesis have been identified as critical factors of aseptic loosening. Although strontium is known to be involved in osteoclast differentiation, its effect on particle-induced inflammatory osteolysis remains unclear. In this study, we investigate the potential impact and underling mechanism of strontium on particle-induced osteoclast activation and chronic inflammation in vivo and in vitro. As expected, strontium significantly inhibited titanium particle-induced inflammatory infiltration and prevented bone loss in a murine calvarial osteolysis model. Interestingly, the number of mature osteoclasts decreased after treatment with strontium in vivo, suggesting osteoclast formation might be inhibited by strontium. Additionally, low receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-α, interleukin-1ß, interleukin-6 and p65 immunochemistry staining were observed in strontium-treatment groups. In vitro, strontium obviously decreased osteoclast formation, osteoclastogenesis-related gene expression, osteoclastic bone resorption and pro-inflammatory cytokine expression in bone-marrow-derived macrophages in a dose-dependent manner. Furthermore, we demonstrated that strontium impaired osteoclastogenesis by blocking RANKL-induced activation of NF-κB pathway. In conclusion, our study demonstrated that strontium can significantly inhibit particle-induced osteoclast activation and inflammatory bone loss by disturbing the NF-κB pathway, and is an effective therapeutic agent for the treatment of wear particle-induced aseptic loosening.

Methylprednisolone promotes recovery of neurological function after spinal cord injury: association with Wnt/ß-catenin signaling pathway activation.

Some studies have indicated that the Wnt/ß-catenin signaling pathway is activated following spinal cord injury, and expression levels of specific proteins, including low-density lipoprotein receptor related protein-6 phosphorylation, ß-catenin, and glycogen synthase kinase-3ß, are significantly altered. We hypothesized that methylprednisolone treatment contributes to functional recovery after spinal cord injury by inhibiting apoptosis and activating the Wnt/ß-catenin signaling pathway. In the current study, 30 mg/kg methylprednisolone was injected into rats with spinal cord injury immediately post-injury and at 1 and 2 days post-injury. Basso, Beattie, and Bresnahan scores showed that methylprednisolone treatment significantly promoted locomotor functional recovery between 2 and 6 weeks post-injury. The number of surviving motor neurons increased, whereas the lesion size significantly decreased following methylprednisolone treatment at 7 days post-injury. Additionally, caspase-3, caspase-9, and Bax protein expression levels and the number of apoptotic cells were reduced at 3 and 7 days post-injury, while Bcl-2 levels at 7 days post-injury were higher in methylprednisolone-treated rats compared with saline-treated rats. At 3 and 7 days post-injury, methylprednisolone up-regulated expression and activation of the Wnt/ß-catenin signaling pathway, including low-density lipoprotein receptor related protein-6 phosphorylation, ß-catenin, and glycogen synthase kinase-3ß phosphorylation. These results indicate that methylprednisolone-induced neuroprotection may correlate with activation of the Wnt/ß-catenin signaling pathway.

Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production.

Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.

Constructing a synthetic metabolic pathway in Escherichia coli to produce the enantiomerically pure (R, R)-2,3-butanediol.

Enantiomerically pure (R, R)-2,3-butanediol has unique applications due to its special chiral group and spatial configuration. Currently, its chemical production route has many limitations. In addition, no native microorganisms can accumulate (R, R)-2,3-butanediol with an enantio-purity over 99%. Herein, we constructed a synthetic metabolic pathway for enantiomerically pure (R, R)-2,3-butanediol biosynthesis in Escherichia coli. The fermentation results suggested that introduction of the synthetic metabolic pathway redistributed the carbon fluxes to the neutral (R, R)-2,3-butanediol, and thus protected the strain against the acetic acid inhibition. Additionally, it showed that the traditionally used isopropyl beta-D-thiogalactoside (IPTG) induction displayed negative effect on (R, R)-2,3-butanediol biosynthesis in the recombinant E. coli, which was probably due to the protein burden. With no IPTG addition, the (R, R)-2,3-butanediol concentration reached 115 g/L by fed-batch culturing of the recombinant E. coli, with an enantio-purity over 99%, which is suitable for the pilot-scale production.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a repeated fed-batch fermentation strategy.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process.

Antitumor efficacy in H22 tumor bearing mice and immunoregulatory activity on RAW 264.7 macrophages of polysaccharides from Talinum triangulare.

The aim of this paper was to study the antitumor and immunoregulatory activities of a polysaccharide (TTP) from Talinum triangulare. The molecular weight of TTP-IV was 49.9 kDa. The monosaccharide composition analysis of TTP-IV revealed that it was a heteropolysaccharide consisting of rhamnose, arabinose, mannose and galactose with a molar ratio of 1.22 : 1.00 : 1.05 : 1.51. The results of the in vivo study showed that TTP (200 mg per kg bw) significantly inhibited the growth of tumor by 49.07% in H22-bearing Kunming mice. In vitro, the growth of primary murine macrophages was promoted by TTP in a dose- and time-dependent manner significantly. Besides, RAW 264.7 cells were activated by TTP to produce NO and the toxicity of RAW 264.7 supernatant was markedly enhanced in vitro. The levels of iNOS, TLR2, TLR4 and IL-1ß were obviously increased by TTP. Therefore, it is suggested that TTP can be utilized as a potent antitumor and immunoenhancing material in functional food.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a three-stage fermentation strategy.

A three-stage fermentation strategy was designed for efficient arachidonic acid (ARA)-rich oil production by Mortierella alpina. The process at different stages by changing the components of medium was investigated. In the first stage, mycelia were inoculated in a nutrient-rich medium for rapid propagation. In the second stage, mycelia were collected and then cultivated in glucose solution to achieve high cellular lipid contents. In the third stage, mycelia were cultured in a glucose-absent medium to obtain rapid ARA accumulation. Using this fermentation strategy, high dry cell weight, lipid, and ARA concentration reached 41.6, 26.6, and 11.4 g/L, respectively. The results demonstrated that mycelia propagation, lipid biosynthesis, and ARA accumulation process can be significantly spatially separated, allowing further optimization to improve the efficiency of each stage. This was the first report of using a three-stage fermentation strategy for ARA-rich oil production, and it could be applied to other similar oleaginous microorganisms to obtain high related polyunsaturated fatty acids accumulation.