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.

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.

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.

Fungal arachidonic acid-rich oil: research, development and industrialization.

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.

Arachidonic acid-rich oil production by Mortierella alpina with different gas distributors.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.

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.

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.

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid.

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l(-1) and 138.8 mg l(-1) h(-1), respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.

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.

Improving arachidonic acid accumulation in Mortierella alpina through B-group vitamin addition.

To improve the arachidonic acid (ARA) accumulation in Mortierella alpina, a mixed B-group vitamin addition strategy was developed. The ARA titer reached up to 10.0 g/L, 1.7-fold of the control. At the same time, the highest specific activities of key enzymes involved in ARA biosynthesis, including malic enzyme, glucose-6-phosphate dehydrogenase and ATP: citrate lyase, were 63.3, 38.6 and 53.7% higher than the control, respectively. The possible vitamin triggered improved ARA accumulation mechanism was thus elucidated that B-group vitamins could function as the cofactors of the key enzymes involved in ARA biosynthesis, or precursors for the formation of NADPH and acetyl-CoA which were crucial for ARA synthesis, and strengthened the related metabolic flux.

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.

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.

Elimination of carbon catabolite repression in Klebsiella oxytoca for efficient 2,3-butanediol production from glucose-xylose mixtures.

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria.

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.

Engineering Klebsiella oxytoca for efficient 2, 3-butanediol production through insertional inactivation of acetaldehyde dehydrogenase gene.

Ethanol was a major byproduct of 2,3-butanediol (2,3-BD) fermentation by Klebsiella oxytoca ME-UD-3. In order to achieve a high efficiency of 2,3-BD production, K. oxytoca mutants deficient in ethanol formation were successfully constructed by replace the aldA gene coding for aldehyde dehydrogenase with a tetracycline resistance cassette. The results suggested that inactivation of aldA led to a significantly improved 2,3-BD production. The carbon flux to 2,3-BD was enhanced by eliminating the byproducing ethanol and at the same time reducing the accumulation of another byproduct acetoin. At last, by fed-batch culturing of the mutant, the final 2,3-BD titer up to 130 g/l with the productivity of 1.63 g/l.h and the 2,3-BD yield relative to glucose of 0.48 g/g was obtained.

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.

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.