Aspergillus niger has shown great potential for hexavalent chromium [Cr(VI)] bioremediation in wastewater treatment. However, the specific functional genes involved in the Cr(VI) removal pathway in A. niger remain unexplored, limiting our ability to create genetically engineered A. niger strains with enhanced bioremediation capabilities. In this study, we successfully identified two critical genes in A. niger responsible for chromate removal: chrR, a putative gene encoding chromate reductase, and chrP, a putative gene encoding chromate permease. Our findings revealed that deletion of either chrR or chrP in A. niger significantly impaired Cr(VI) removal. Conversely, overexpressing chrR or chrP increased Cr(VI) removal rates by 34.0% and 29.7%, respectively, compared to the parental strain. Additionally, our study showed that sodA, one of the three cytoplasmic superoxide dismutase genes in A. niger, plays a pivotal role in detoxifying reactive oxygen species (ROS) stress induced by Cr(IV). Furthermore, by cooverexpressing chrR, chrP, and sodA to simultaneously enhance the Cr(VI) uptake, reduction, and ROS detoxification pathway, we successfully engineered an A. niger strain that exhibited a remarkable 92% improvement in Cr(VI) removal compared to the wild-type strain. This achievement highlights the significant potential of genetic engineering in addressing Cr(VI) pollution through bioremediation.

Because of the limited effectiveness of prevailing phylogenetic methods when applied to highly divergent protein sequences, the phylogenetic analysis problem remains challenging. Here, we propose a sequence-based evolutionary distance algorithm termed sequence distance (SD), which innovatively incorporates site-to-site correlation within protein sequences into the distance estimation. In protein superfamilies, SD can effectively distinguish evolutionary relationships both within and between protein families, producing phylogenetic trees that closely align with those based on structural information, even with sequence identity less than 20%. SD is highly correlated with the similarity of the protein structure, and can calculate evolutionary distances for thousands of protein pairs within seconds using a single CPU, which is significantly faster than most protein structure prediction methods that demand high computational resources and long run times. The development of SD will significantly advance phylogenetics, providing researchers with a more accurate and reliable tool for exploring evolutionary relationships.

Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

Background Kojic acid (KA) is a widely used compound in the cosmetic, medical, and food industries, and is typically produced by Aspergillus oryzae . To meet increasing market demand, it is important to optimize KA production through seeking alternatives that are more economic than current A. oryzae -based methods. Results In this study, we achieved the first successful heterologous production of KA in Aspergillus niger , an industrially important fungus that does not naturally produce KA, through the expression of the kojA gene from A. oryzae . Using the resulting KA-producing A. niger strain as a platform, we identified four genes ( nrkA , nrkB , nrkC , and nrkD ) that negatively regulate KA production. Knocking down nrkA or deleting any of the other three genes resulted in a significant increase in KA production in shaking flask cultivation. The highest KA titer (25.71 g/L) was achieved in a pH controlled batch bioreactor using the kojA overexpression strain with a deletion of nrkC , which showed a 26.7% improvement compared to the KA titer (20.29 g/L) that was achieved in shaking flask cultivation. Conclusion Our study demonstrates the potential of using A. niger as a platform for studying KA biosynthesis and regulation, and for the cost-effective production of KA in industrial strain development.

Dacarbazine (DTIC) dominates chemotherapy for malignant melanoma (MM). However, the hydrophobicity, photosensitivity, instability, and toxicity to normal cells of DTIC limit its efficacy in treating MM. In the present study, we constructed star-shaped block polymers nanoparticles (NPs) based on Cholic acid -poly (lactide-co-glycolide)-b-polyethylene glycol (CA-PLGA-b-PEG) for DTIC encapsulation and MM targeted therapy. DTIC-loaded CA-PLGA-b-PEG NPs (DTIC-NPs) were employed to increase the drug loading and achieve control release of DTIC, followed by further modification with nucleic acid aptamer AS1411 (DTIC-NPs-Apt), which played an important role for active targeted therapy of MM. In vitro, DTIC-NPs-Apt showed good pH-responsive release and the strongest cytotoxicity to A875 cells compared with DTIC-NPs and free DTIC. In vivo results demonstrated that the versatile DTIC-NPs-Apt can actively target the site of MM and exhibited excellent anti-tumor effects with no obvious side effects. Overall, this research provided multi-functional NPs, which endow a new option for the treatment of MM.

Background Itaconic acid (IA) is a versatile platform chemical widely used for the synthesis of various polymers and current methods for IA production based on Aspergillus terreus fermentation are limited in terms of process efficiency and productivity. To construct more efficient IA production strains, A. niger was used as a chassis for engineering IA production by assembling the key components of IA biosynthesis pathways from both A. terreus and Ustilago maydis . Results Recombinant A. niger S1596 overexpressing the A. terreus IA biosynthesis genes cadA , mttA , mfsA produced IA of 4.32 g/L, while A. niger S2120 overexpressing the U. maydis IA gene cluster adi1 , tad1 , mtt1 , itp1 achieved IA of 3.02 g/L. Integration of the two IA production pathways led to the construction of A. niger S2083 with IA titers of 5.58 g/L. Increasing cadA copy number in strain S2083 created strain S2209 with titers of 7.99 g/L and deleting ictA to block IA degradation in S2209 created strain S2288 with IA titers of 8.70 g/L. Overexpressing acoA to enhance the supply of IA precursor in strain S2288 generated strain S2444 with IA titers of 9.08 g/L in shake flask. Conclusion Recombinant A. niger overexpressing the U. maydis IA biosynthesis pathway was capable of IA accumulation. Combined expression of the two IA biosynthesis pathways from A. terreus and U. maydis in A. niger resulted in much higher IA titers . Furthermore, increasing cadA copy number, deleting ictA to block IA degradation and overexpressing acoA to enhance IA precursor supply all showed beneficial effects on IA accumulation.

Aspergillus niger is a major cell factory for citric acid production, and the process of citrate export from mitochondria to cytoplasm is predicted to be one of rate-limiting steps in citric acid accumulation. Currently, the mitochondrial citrate transporters (Ctps) in A. niger are not fully characterized. Here, six putative Ctp encoding genes (ctpA to ctpF) were identified based on their homology with a mitochondrial citrate transporter ScCtp1 from Saccharomyces cerevisiae. Disruption of individual ctpA to ctpF caused varying degrees of decline in citric acid accumulation at different fermentation stages, whereas a mutant strain S1696 with disruption of all six ctps showed complete loss of citiric acid production. S1696 also exhibited delayed growth, reduced conidia formation, and decreased pigmentogenesis. Exogenous addition of citrate partially restored the conidia formation and pigmentogenesis in S1696 mutant. Reintroduction of individual ctps (ctpA to ctpF) into S1696 at the amyA locus showed that ctpA, ctpB, and ctpD restored the citric acid titers to 88.5, 93.8, and 94.6% of the parent strain, respectively. Additionally, the formation of conidia and pigment production was partially restored after reintroduction of ctpA, ctpB, or ctpD. Overexpression of respective ctpA, ctpB, and ctpD in the parent strain resulted in increases in citric acid accumulation by 32.8, 19.3, and 24.2%, respectively. These results demonstrate that CtpA, CtpB, and CtpD play important roles in citric acid transport across the mitochondrial membrane and function in a redundant manner. Enhancement of citric acid transport process can serve as a target for boosting citric acid accumulation in A. niger.

FtsH belongs to the AAA+ ATP-dependent family of proteases, which participate in diverse cellular processes and are ubiquitous among bacteria, chloroplasts, and mitochondria. FtsH is poorly characterized in most organisms, especially compared to other major housekeeping proteases. In the current study, we examined the source of FtsH essentiality in the human oral microbiome species Streptococcus mutans, one of the primary etiological agents of dental caries. By creating a conditionally lethal ftsH mutant, we were able to identify a secondary suppressor missense mutation in the vicR gene, encoding the response regulator of the essential VicRK two-component system (TCS). Transcriptomic analysis of the vicR (G195R) mutant revealed significantly reduced expression of 46 genes, many of which were located within the genomic island Tnsmu2, which harbors the mutanobactin biosynthetic gene cluster. In agreement with the transcriptomic data, deletion of the mutanobactin biosynthetic gene cluster suppressed ftsH essentiality in S. mutans. We also explored the role of FtsH in S. mutans physiology and demonstrated its critical role in stress tolerance, especially acid stress. The presented results reveal the first insights within S. mutans for the pleiotropic regulatory function of this poorly understood global regulator.

Modification of C4-dicarboxylate transport processes is an important strategy for the development of efficient malic acid producing cell factory in Aspergillus niger. However, there is a lack of identification and functional research of malic acid transport proteins, which seriously hinders the construction of high-yield malic acid metabolic engineering strains. A C4-dicarboxylate transport protein (DCT) DCT1 is identified as major malic acid transport protein and exhibits significant elevation in malic acid production when overexpressed. DCT1 is found by homology searches and domain analyses with SpMAE1 from Schizosaccharomyces pombe as the template. Phylogenetic and domain analyses show that DCTs belong to voltage-dependent slow-anion channel transporter (SLAC1) family and are members of Tellurite-resistance/Dicarboxylate Transporter (TDT) Family. DCT1 disruption dramatically decreases malic acid titer by about 85.6% and 96.2% at 3 days and 5 days compared with the parent strain, respectively. Meanwhile, the citric acid titers increase by 36.4% and 13.7% at 3 days and 5 days upon DCT1 deficiency. These results suggest that DCT1 is the major malic acid transporter in A. niger. Overexpression of dct1 with its native promoter significantly improves malic acid production yielding up to 13.86 g/L and 30.79 g/L at 3 days and 5 days, respectively, which is 36.8% and 22.8% higher than those in the parent strain. However, the citric acid has no significant change during the 5-day fermentation. These results demonstrate the importance of C4-dicarboxylate transporters for the efficient production of malic acid. Furthermore, enhancement of malic acid transport process is a feasible approach of efficient malic acid production in this citric acid producing A. niger strain. • A dicarboxylate transporter DCT1 is identified as a major malic acid transporter. • DCT1 deficiency results in significant decrease of malic acid. • DCT1 overexpression leads to increased titers of malic acid. • Enhancement of malic acid transport is vital for malic acid production in A. niger.

Fluoride has been used as an effective anticaries agent for more than 70 years, which might result in the emergence of fluoride-resistant strains. However, the fluoride resistance mechanism and the cariogenic properties of fluoride-resistant mutant for cariogenic bacterial speciesStreptococcus mutansremain largely unknown. We describe here the construction and characterization of a mariner-based transposon system designed to be used inS. mutans, which is also potentially applicable to other streptococci. To identify genetic determinants of fluoride resistance inS. mutans, we constructed a library ofS. mutanstransposon insertion mutants and screened this library to identify mutants exhibiting fluoride resistance phenotype. Two mutants were found to carry transposon insertion in two different genetic loci (smu.396andsmu.1291c), respectively. Our subsequent genetic study indicates the fluoride-resistant phenotype for the mutant with the insertion insmu.1291cis resulting from the constitutive overexpression of downstream operonsmu.1290c-89c, which is consistent with the previous reports. We also demonstrate for the first time that the deletion ofsmu.396is responsible for the fluoride-resistant phenotype and that the combining ofsmu1290c-89coverexpression andsmu.396deletion in one strain could attribute an additive effect on the fluoride resistance. In addition, our results suggest that the biological fitness of those fluoride-resistant mutants is reduced compared to that of wild-type strain. Overall, our identification and characterization of genetic determinants responsible for fluoride resistance inS. mutansexpand our understanding of the fluoride resistance mechanism and the biological consequence of the fluoride resistance strains.

Microbial fermentation was widely explored to produce malic acid. Previously, Aspergillus niger has been successfully engineered, and a high titer of malic acid was achieved with strain S575, but it also produced a high level of byproduct citric acid. Here, the capability of A. niger in malic acid biosynthesis was further improved by eliminating the accumulation of citric acid and enhancing glycolytic flux. Characterization of variant mutants suggested that disruption of cexA, a gene encoding citric acid transporter located on cell membrane, abolished citric acid accumulation. However, cexA-deficient strain S895 showed significantly decreased malic acid production. Further analysis of S895 indicated that the transcription level of genes involved in glucose transportation and glycolytic pathway was significantly reduced, and the corresponding enzyme activity was also lower than those of S575. Individual overexpression of genes encoding glucose transporter MstC and key enzymes (hexokinase HxkA, 6-phosphofructo-2-kinase PfkA, and pyruvate kinase PkiA) involved in irreversible reactions of glycolic pathway increased malic acid production. Accordingly, genes of mstC, hxkA, pfkA, and pkiA were overexpressed altogether in S895, and the resultant strain S1149 was constructed. The titer of malic acid in fed-batch fermentation with S1149 reached 201.13 g/L. Compared with S575, the byproduct of citric acid was completely abolished in S1149, and the ratio of malic acid/glucose was increased from 1.27 to 1.64 mol/mol, the highest yield reported so far, and the fermentation period was shortened from 9 to 8 days. Thus, a strain with great industrial application potential was developed by engineering nine genes in A. niger, and a pilot fermentation technology was exploited.

黑曲霉是一种重要的发酵工业微生物,也是用于研究丝状真菌生长、发育、代谢、基因功能等的模式微生物.将构巢曲霉来源的乙酰胺酶编码基因amdS在黑曲霉中异源表达,成功得到黑曲霉amdS转化子.经表型分析,amdS赋予了黑曲霉在乙酰胺为唯一氮源的培养基上生长的能力,而野生型菌株则生长微弱.构建了以amdS为筛选标记的基因敲除载体,以草酰乙酸水解酶基因oahA上下游同源序列构建oahA敲除质粒,转化黑曲霉后成功筛选到oahA敲除菌株,草酸合成水平明显降低.结果 表明,利用amdS作为营养筛选标记能够很好地应用于黑曲霉遗传转化操作中.

The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre- lox P-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter P xln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of lox P site-specific recombination in the strain with cre driven by P xln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase–encoding gene ( oahA ) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation ( oahA deletion, pyc , mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre- lox P system is a powerful tool for genetic engineering in A. niger , which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.

Streptococcus zooepidemicus is an important opportunistic pathogen of several species including humans. This organism is also well known as the main producing strain in industrial production of hyaluronic acid (HA), which is the component of its capsule polysaccharide. How its virulence and capsule polysaccharide production is regulated remains poorly understood. Intercellular chemical signaling among bacteria provides communities of microbes the opportunity to coordinate gene expression to facilitate group behavior, such as pathogenicity, capsule polysaccharide production, etc. Yet no conserved cell-to-cell signaling system has been elucidated in S. zooepidemicus. Encoded within the genome of S. zooepidemicus is one Rgg regulator encoding gene (rgg) with low similarity to both rgg2 and rgg3 from Streptococcus pyogenes. A small ORF (named as shp) encoding a novel short hydrophobic peptide (SHP) was found in the vicinity of rgg. We found that the active form of pheromone is short and hydrophobic (LLLLKLA), corresponding to the C terminal 7 amino acids of the pre-peptide Shp, which shows divergent sequence to all peptide pheromones reported in streptococci. In response to active SHP, Rgg functions as a transcriptional activator to induce the expression of shp, forming a positive feedback circuit. Bacteria social behaviors, such as capsule polysaccharide production and biofilm formation, were significantly affected when the rgg-shp pathway was inactivated. These data provide the first demonstration that Rgg/Shp signaling pathway comprises an active quorum sensing system in S. zooepidemicus.

To evaluate a rapid and simple technique for determining viability of 11 strains of marine mi-croalgae (10～50 μm)in the ballast water,three staining dyes (Neutral Red (NR),Fluorescein Diacetate (FDA)and 5-Chloromethylfluorescein Diacetate (CMFDA))were applied to the living cells and heat killed cells of microalgae.The result showed that NR was the best dye for determning the living cells of all ma-rine microalgae species tested in the study.The optimum staining concentration was 1/10000,and the staining time was 30 min.CMFDA,FDA and CMFDA+FDA were also available with short staining time, obvions staining effect,but limited application in this study,only suitble for cells of P latymonas helgo-landica var.tsingtaoensis ,Dunaliella salina ,Prorocentrum donghaiense ,Karenia mikimotoi ,Proro-centrum minimum ,Alexandrium catenella ,Alexandrium tamarense ,and Prorocentrum lima .The opti-mum staining concentration was FDA (5 μmol/L)+ CMFDA (2.5 μmol/L),and the staining time was 10 min.Based on the results,NR staining method is more suitable for detecting the microalgae cell viability in ballast water.

Triple-negative breast cancers (TNBCs) are the most aggressive and hard-to-treat breast tumors with poor prognosis, and exploration for novel therapeutic drugs is impending. Arctigenin (Atn), a bioactive lignan isolated from seeds of Arctium lappa L, has been reported to inhibit many cancer types; however, the effect of Atn on TNBC remains unclear. In this study, we demonstrated that Atn decreased proliferation, and induced apoptosis in TNBC cells. Furthermore, we explored the underlying mechanism of Atn inhibition on TNBC cells. Computational docking and affinity assay showed that Atn bound to the SH2 domain of STAT3. Atn inhibited STAT3 binding to genomic DNA by disrupting hydrogen bond linking between DNA and STAT3. In addition, Atn augmented Taxotere®-induced TNBC cell cytotoxicity. TNBC xenograft tests also confirmed the antitumor effect of Atn in vivo. These characteristics render Atn as a promising candidate drug for further development and for designing new effective STAT3 inhibitors.

Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous protein phosphatase 2A (PP2A) inhibitor, has been identified as an oncoprotein in promoting cancer initiation and progression of several types of cancer. However, the expression and the role played by CIP2A in the pathogenesis of multiple myeloma (MM) remain unclear. In this study, we showed that CIP2A was overexpressed in human MM cell lines and MM patients’ bone marrow tissues. Clinicopathologic analysis showed that CIP2A expression was significantly correlated with clinical stage and percent of plasma cells in bone marrow. Kaplan–Meier analysis revealed that patients with high CIP2A expression presented with poorer overall survival rates than those with low CIP2A expression. Moreover, CIP2A knockdown in MM cells resulted in attenuated proliferative abilities. In addition, CIP2A depletion sensitizes dexamethasone (Dex)-resistant cells to Dex. The effect of CIP2A on proliferation and Dex therapy was mediated by the inhibition of PP2A, which in turn activated Akt. In vivo studies confirmed that CIP2A regulated MM tumorigenesis and the phosphorylation of Akt. Taken together, our results suggest that CIP2A oncoprotein plays an important role in MM progression and could serve as a prognosis marker and a novel therapeutic target for the treatment of patients with MM.

Oridonin (Ori), a diterpenoid compound extracted from traditional medicinal herbs, elicits antitumor effects on many cancer types. However, whether Ori can be used in gefitinib-resistant non-small cell lung cancer (NSCLC) cells remains unclear. This study investigated the antitumor activity and underlying mechanisms of Ori. Results demonstrated that this compound dose-dependently inhibited the proliferation, invasion, and migration of the gefitinib-resistant NSCLC cells in vitro. Ori also significantly downregulated the phosphorylation of EGFR, ERK, Akt, expression levels of matrix metalloproteinase-12 (MMP-12), and the cancerous inhibitor of protein phosphatase 2A (CIP2A). In addition, Ori upregulated protein phosphatase 2A (PP2A) activity of gefitinib-resistant NSCLC cells. Ori combined with docetaxel synergistically inhibited these cells. Ori also inhibited tumor growth in murine models. Immunohistochemistry results further revealed that Ori downregulated phospho-EGFR, MMP-12, and CIP2A in vivo. These findings indicated that Ori can inhibit the proliferation, invasion, and migration of gefitinib-resistant NSCLC cells by suppressing EGFR/ERK/MMP-12 and CIP2A/PP2A/Akt signaling pathways. Thus, Ori may be a novel effective candidate to treat gefitinib-resistant NSCLC.

Ultraviolet (UV) radiation has deleterious effects on living organisms, and functions as a tumor initiator and promoter. Multiple natural compounds, like quercetin, have been shown the protective effects on UV-induced damage. However, quercetin is extremely hydrophobic and limited by its poor percutaneous permeation and skin deposition. Here, we show that quercetin-loaded PLGA-TPGS nanoparticles could overcome low hydrophilicity of quercetin and improve its anti-UVB effect. Quercetin-loaded NPs can significantly block UVB irradiation induced COX-2 up-expression and NF-kB activation in Hacat cell line. Moreover, PLGA-TPGS NPs could efficiently get through epidermis and reach dermis. Treatment of mice with quercetin-loaded NPs also attenuates UVB irradiation-associated macroscopic and histopathological changes in mice skin. These results demonstrated that copolymer PLGA-TPGS could be used as drug nanocarriers against skin damage and disease. The findings provide an external use of PLGA-TPGS nanocarriers for application in the treatment of skin diseases. (C) 2015 Elsevier Inc. All rights reserved.

PICT-1 is an essential ribosome biogenesis factor whose loss induces p53 accumulation and apoptosis. Here, we show that DNA damage changes PICT-1 localization and decreases PICT-1 protein levels via the proteasome pathway. Two important phosphatidylinositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and the Ku70 subunit of DNA-dependent protein kinase (DNA-PK), co-localize and interact with PICT-1 in the nucleolus. Computational prediction of phosphorylation sites and detection using an anti-phospho-substrate antibody suggest that PICT-1 might be a substrate of PIKKs. PICT-1 S233 and T289 were identified as the key phosphorylation sites in this pathway, as mutating both to alanine abolished UVB-induced increase of PICT-1 phosporylation. Inhibition of PIKKs or ATM (with wortmannin and KU55933, respectively) prevented the agglomeration and degradation of PICT-1, suggesting that ATM is a key regulator of PICT-1. PICT-1(S233A, T289A) demonstrated marked resistance to DNA damage-induced agglomeration and loss of PICT-1. Phosphomimetic PICT-1 (S233D, T289D) showed a different nuclear distribution and was more rapidly degraded after DNA damage than wild-type PICT-1. Furthermore, both phosphorylation and degradation of PICT-1 released RPL11 from the nucleolus to the nucleoplasm, increased binding of RPL11 to MDM2, and promoted p53 accumulation and apoptosis in an ATM-dependent manner after DNA damage. These data indicate that PICT-1 is a major nucleolar sensor of the DNA damage repair response and an important upstream regulator of p53 via the RPL11-MDM2-p53 pathway.