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Corynebacterium glutamicum represents an emerging recombinant protein expression factory due to its ideal features for protein secretion, but its applicability is harmed by the lack of an autoinduction system with tight regulation and high yield. Here, we propose a new recombinant protein manufacturing platform that leverages ethanol as both a delayed carbon source and an inducer. First, we reanalysed the native inducible promoter PICL from the acetate uptake operon and found that its limited capacity is the result of the inadequate translation initial architecture. The two strategies of bicistronic design and ribozyme-based insulator can ensure the high activity of this promoter. Next, through transcriptional engineering that alters transcription factor binding sites (TFBSs) and the first transcribed sequence, the truncated promoter PA256 with a dramatically higher transcription level was generated. When producing the superfolder green fluorescent protein (sfGFP) under 1% ethanol conditions, PA256 exhibited substantially lower protein accumulation in prophase but an approximately 2.5-fold greater final yield than the strong promoter PH36. This superior expression mode was further validated using two secreted proteins, camelid antibody fragment (VHH) and endoxylanase (XynA). Furthermore, utilizing CRISPRi technology, ethanol utilization blocking strains were created, and PA256 was shown to be impaired in the phosphotransacetylase (PTA) knockdown strains, indicating that ethanol metabolism into the tricarboxylic acid cycle is required for PA256 upregulation. Finally, this platform was applied to produce the "de novo design" protein NEO-2/15, and by introducing the N-propeptide of CspB, NEO-2/15 was effectively secreted with the accumulation 281 mg/L obtained after 24 h of shake-flask fermentation. To the best of our knowledge, this is the first report of NEO-2/15 secretory overexpression. [ABSTRACT FROM AUTHOR]

Our previous acute study on urocortin (Ucn) demonstrated that Ucn altered serum and tissue angiotensin-converting enzyme (ACE) activity in rats. Therefore, the present investigation was designed to explore the effect of long-term treatment with Ucn on somatic ACE (sACE) and other components of the renin-angiotensin system (RAS). After 8 weeks of intravenous administration of Ucn in spontaneously hypertensive rats (SHR), serum and tissue sACE, angiotensin II (Ang II), nitric oxide (NO), Ang-(1-7), and tissue chymase activities were evaluated. RT-PCR analysis was performed to determine the quantity of tissue sACE mRNA. Serum sACE activity was reduced by Ucn, although tissue sACE activity and tissue sACE mRNA were elevated. Chymase activity was observed to be enhanced by Ucn, whereas the ACE inhibitor enalapril failed to influence chymase. Serum and tissue Ang II activity was reduced, but NO and Ang-(1-7) production was increased in a concentration-dependent manner after Ucn treatment. Meanwhile, a significant decrease of the systolic blood pressure (SBP) was observed after the long-term Ucn administration, and there was a significant positive correlation (r2 = 0.6993) between serum ACE activity and SBP. Pretreatment with the corticotropin-releasing factor (CRF) blocker astressin and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway blocker PD98059 abolished these effects of Ucn. Our findings further support the hypothesis that the changes of sACE activity and the production of other RAS components may play roles in the vasodilatory property of Ucn via the activation of the ERK1/2 pathway. [ABSTRACT FROM AUTHOR]

Oxidative stress contributes to the pathogenesis of diabetic nephropathy (DN). Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in cellular defense against oxidative stress. NRF2 activators have shown promising preventive effects on DN. Sodium butyrate (NaB) is a known activator of NRF2. However, it is unknown whether NRF2 is required for NaB protection against DN. Therefore, streptozotocin-induced diabetic C57BL/6 Nrf2 knockout and their wild-type mice were treated in the presence or absence of NaB for 20 weeks. Diabetic mice, but not NaB-treated diabetic mice, developed significant renal oxidative damage, inflammation, apoptosis, fibrosis, pathological changes and albuminuria. NaB inhibited histone deacetylase (HDAC) activity and elevated the expression of Nrf2 and its downstream targets heme oxygenase 1 and NAD(P)H dehydrogenase quinone 1. Notably, deletion of the Nrf2 gene completely abolished NaB activation of NRF2 signaling and protection against diabetes-induced renal injury. Interestingly, the expression of Kelch-like ECH-associated protein 1, the negative regulator of NRF2, was not altered by NaB under both diabetic and nondiabetic conditions. Moreover, NRF2 nuclear translocation was not promoted by NaB. Therefore, the present study indicates, for the first time, that NRF2 plays a key role in NaB protection against DN. Other findings suggest that NaB may activate Nrf2 at the transcriptional level, possibly by the inhibition of HDAC activity. [ABSTRACT FROM AUTHOR]

Apple ring rot, which is caused by Botryosphaeria dothidea, severely affects apple production. The mechanisms employed in apple cells against B. dothidea remain unknown. In this research, the pathogen infection mode and the relationship between cell death and disease resistance in 'Fuji'/B. dothidea interaction pathosystem were investigated. By using transmission electron microscopy (TEM), our research showed that the pathogen infects apple cells both intracellularly and extracellularly. However, compared with that in immature fruit, the incidence of hyphae in the interior of mature apple fruit cells increased dramatically, suggesting that cell wall-mediated penetration resistance could be important in apple resistance against B. dothidea. TEM ultrastructural characterization identified the nuclear morphology of programmed cell death induction in both apple fruit and callus cells under B. dothidea infection. Overexpression of MdVDAC2 (MDP0000271281), which encodes an outer-membrane localized anion channel protein in mitochondria, significantly promoted cell death under B. dothidea infection and simultaneously inhibited pathogen infection, suggesting that cell death represents a disease resistance mechanism in apple against B. dothidea infection. Furthermore, BdCatalase (KAF4307763), a cytochrome P450 family protein BdCYP52A4 (KAF4300696), and subtilisin-domain containing proteins were identified from B. dothidea -secreted proteins, which suggested the potential involvement of active oxygen species and phytoalexins in combating B. dothidea infection and triggering or dampening apple resistance. Collectively, our research suggested that cell wall-mediated penetration resistance, programmed cell death machinery and microbial effector-interrelated signaling were among strategies recruited in apple to combat B. dothidea. The current research laid the foundation for further investigations into resistance mechanisms in apple. [ABSTRACT FROM AUTHOR]

Cyclin-dependent Kinase 2 (CDK2) inhibition prevents supernumerary centrosome clustering. This causes multipolarity, anaphase catastrophe and apoptotic death of aneuploid cancers. This study elucidated how CDK2 antagonism affected centrosome stoichiometry. Focused ion beam scanning electron microscopy (FIB-SEM) and immunofluorescent imaging were used. Studies interrogated multipolar mitosis after pharmacologic or genetic repression of CDK2. CDK2/9 antagonism with CYC065 (Fadraciclib)- treatment disordered centrosome stoichiometry in aneuploid cancer cells, preventing centrosome clustering. This caused ring-like chromosomes or multipolar cancer cells to form before onset of cell death. Intriguingly, CDK2 inhibition caused a statistically significant increase in single centrioles rather than intact centrosomes with two centrioles in cancer cells having chromosome rings or multipolarity. Statistically significant alterations in centrosome stoichiometry were undetected in other mitotic cancer cells. To confirm this pharmacodynamic effect, CDK2 but not CDK9 siRNA-mediated knockdown augmented cancer cells with chromosome ring or multipolarity formation. Notably, engineered gain of CDK2, but not CDK9 expression, reversed emergence of cancer cells with chromosome rings or multipolarity, despite CYC065-treatment. In marked contrast, CDK2 inhibition of primary human alveolar epithelial cells did not confer statistically significant increases of cells with ring-like chromosomes or multipolarity. Hence, CDK2 antagonism caused differential effects in malignant versus normal alveolar epithelial cells. Translational relevance was confirmed by CYC065-treatment of syngeneic lung cancers in mice. Mitotic figures in tumors exhibited chromosome rings or multipolarity. Thus, CDK2 inhibition preferentially disorders centrosome stoichiometry in cancer cells. Engaging this disruption is a strategy to explore against aneuploid cancers in future clinical trials. [ABSTRACT FROM AUTHOR]

Real-time quantitative PCR (qRT-PCR) is a fast and efficient technology for detecting gene expression levels in the study of the Corynebacterium glutamicum protein expression system, but it requires normalization to ensure the reliability of the results obtained. We selected 13 genes from the commonly used housekeeping genes and from transcriptome data as candidate reference genes. The Ct values of the 13 genes were obtained by qRT-PCR at different fermentation stages and under three stress conditions (temperature, acid and salt). The expression stability of the reference genes was evaluated by geNorm and NormFinder software. For the study of different growth stages, the most appropriate reference genes are Ncgl2772 and leua, which encode acetyl-CoA carboxylase beta subunit and 2-isopropylmalate synthase, separately. For the study of different stress factors, the optimal minimum number of reference genes is 3, with Ncgl2772, gyrb encoding DNA gyrase B and siga encoding RNA polymerase sigma factor A as the most suitable combination. Additionally, clpx and clpc, encoding ClpX and ClpC protease subunits, were used to validate the candidate reference genes. The identification of new reference genes makes qRT-PCR more convenient, and using these genes for normalization can improve the accuracy and reliability of the measurements of target gene expression levels obtained by qRT-PCR for C. glutamicum. [ABSTRACT FROM AUTHOR]

The efficient promoter of alcohol oxidase 1 (PAOX1) in methylotrophic yeast Pichia pastoris is strictly induced by methanol but repressed by glycerol with an unclear molecular mechanism. In the present study, the gene of a previously characterized transmembrane protein glycerol transporter 1 (GT1) of P. pastoris GS115 was deleted by homologous recombination. Transcriptional profiles of the mutant (gt1Δ) and wild type (WT) were compared with different carbon sources (glycerol, methanol and glycerol-methanol mix) at various time points using high-throughput RNA-Seq techniques. We determined that the loss of glycerol transporter 1 (Gt1p) could relieve catabolite repression in the glycerol-methanol mixed medium and shared a similar transcriptional profile with the WT in methanol medium. By calculating the common differentially expressed genes in three distinct paired groups, genes involved in the stress response, nutrition deprivation and translational process were identified, explaining the potential roles of glycerol in the regulation of methanol metabolism. Based on weighted gene co-expression network analysis, the relationship between biological traits and the transcriptional profile was established. With the support of published research and our data, we propose two possible regulatory pathways that are involved in the regulation of catabolite repression (adenosine 5'-monophosphate (AMP)-activated protein kinase /SNF1 and Mitogen-activated protein kinase/HOG), thereby providing potential targets for both research and industrial strain improvement. [ABSTRACT FROM AUTHOR]

Background: Corynebacterium glutamicum (C. glutamicum) has traditionally been used as a microbial cell factory for the industrial production of many amino acids and other industrially important commodities. C. glutamicum has recently been established as a host for recombinant protein expression; however, some intrinsic disadvantages could be improved by genetic modification. Gene editing techniques, such as deletion, insertion, or replacement, are important tools for modifying chromosomes. Results: In this research, we report a CRISPR/Cas9 system in C. glutamicum for rapid and efficient genome editing, including gene deletion and insertion. The system consists of two plasmids: one containing a target-specific guide RNA and a homologous sequence to a target gene, the other expressing Cas9 protein. With high efficiency (up to 100%), this system was used to disrupt the porB, mepA, clpX and Ncgl0911 genes, which afect the ability to express proteins. The porB- and mepA-deletion strains had enhanced expression of green fluorescent protein, compared with the wild-type stain. This system can also be used to engineer point mutations and gene insertions. Conclusions: In this study, we adapted the CRISPR/Cas9 system from S. pyogens to gene deletion, point mutations and insertion in C. glutamicum. Compared with published genome modification methods, methods based on the CRISPR/Cas9 system can rapidly and efficiently achieve genome editing. Our research provides a powerful tool for facilitating the study of gene function, metabolic pathways, and enhanced productivity in C. glutamicum. [ABSTRACT FROM AUTHOR]

In the methylotrophic yeast Pichia pastoris (P. pastoris), the efficient promoter of alcohol oxidase (PAox1) is induced by methanol and repressed by glycerol, but the molecular mechanism is not clear. In this study, the relationship between alcohol oxidase 1 (aox1), methanol expression regulator 1 (mxr1) and glycerol transporter 1 (gt1) was studied. By RT-PCR, it was found that the overexpression of gt1 could increase the glycerol content in cells and repress the expression of mxr1 and aox1, and the deletion of gt1 reduced the glycerol content in cells and promoted the expression of aox1. The overexpression of mxr1 could repress the expression of gt1, and the deletion of mxr1 could promote the expression of gt1 to some extent. By EMSA, Mxr1 binding sites were found in the promoter of gt1 (PGt1) (-141 to -138, CCCC), and Mxr1 could regulate the expression of gt1 by binding to PGt1. The relationships among aox1, mxr1 and gt1 revealed here provide a reference for the understanding of the mechanism of glycerol repression of PAox1. [ABSTRACT FROM AUTHOR]

Promoter of alcohol oxidase I (PAOX1) is the most efficient promoter involved in the regulation of recombinant protein expression in Pichia pastoris (P. pastoris). PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of the heterologous protein can be induced. In this study, a candidate glycerol transporter (GT1, GeneID: 8197545) was identified, and its role was confirmed by further studies (e.g. bioinformatics analysis, heterologous complementation in Schizosaccharomyces pombe (S. pombe)). When GT1 is co-expressed with enhanced green fluorescent protein (EGFP), it localizes to the membrane and S. pombe carrying gt1 but not the wild-type strain can grow on medium containing glycerol as the sole carbon source. The present study is the first to report that AOX1 in the X-33⧠gt1 mutant can achieve constitutive expression in medium containing glycerol; thus, knocking down gt1 can eliminate the glycerol repression of PAOX1 in P. pastoris. These results suggest that the glycerol transporter may participate in the process of PAOX1 inhibition in glycerol medium. [ABSTRACT FROM AUTHOR]

The ubiquitin-like protein FAT10 and the homeobox protein HOXB9 each promote metastatic progression in hepatocellular carcinoma (HCC). In this study, we investigated the clinicopathologic significance of FAT10 and HOXB9 in HCC and investigated a mechanistic role for FAT10 in HOXB9-mediated invasiveness and metastasis. Relative to adjacent normal tissues, FAT10 and HOXB9 were markedly overexpressed in HCC, where a positive correlation in their expression and associated malignant characteristics were found. RNAi-mediated silencing of FAT10 decreased HOXB9 expression and inhibited HCC invasion and metastasis in vitro and in vivo. The effects of FAT10 silencing were reversed by HOXB9 overexpression, whereas RNAi-mediated silencing of HOXB9 decreased HCC invasion and metastasis driven by FAT10 overexpression. Mechanistically, FAT10 regulated HOXB9 expression by modulating the β-catenin/TCF4 pathway, directly binding to β-catenin and preventing its ubiquitination and degradation. Together, our results identified a novel HCC regulatory circuit involving FAT10, β-catenin/TCF4, and HOXB9, the dysfunction of which drives invasive and metastatic character in HCC. [ABSTRACT FROM AUTHOR]