Harnessing native-cryptic plasmids for stable overexpression of heterologous genes in Clostridium butyricum DSM 10702 for industrial and medical applications.

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum.

Assessment of CRISPRa-mediated gdnf overexpression in an In vitro Parkinson's disease model.

Introduction: Parkinson's disease (PD) presents a significant challenge in medical science, as current treatments are limited to symptom management and often carry significant side effects. Our study introduces an innovative approach to evaluate the effects of gdnf overexpression mediated by CRISPRa in an in vitro model of Parkinson's disease. The expression of gdnf can have neuroprotective effects, being related to the modulation of neuroinflammation and pathways associated with cell survival, differentiation, and growth. Methods: We have developed a targeted delivery system using a magnetite nanostructured vehicle for the efficient transport of genetic material. This system has resulted in a substantial increase, up to 200-fold) in gdnf expression in an In vitro model of Parkinson's disease using a mixed primary culture of astrocytes, neurons, and microglia. Results and Discussion: The delivery system exhibits significant endosomal escape of more than 56%, crucial for the effective delivery and activation of the genetic material within cells. The increased gdnf expression correlates with a notable reduction in MAO-B complex activity, reaching basal values of 14.8 µU/µg of protein, and a reduction in reactive oxygen species. Additionally, there is up to a 34.6% increase in cell viability in an In vitro Parkinson's disease model treated with the neurotoxin MPTP. Our study shows that increasing gdnf expression can remediate some of the cellular symptoms associated with Parkinson's disease in an in vitro model of the disease using a novel nanostructured delivery system.

The quality and flavor profile of fermented milk produced by Streptococcus thermophilus ABT-T is influenced by the pfs gene in the quorum sensing system.

The luxS/AI-2 quorum sensing (QS) system of Streptococcus thermophilus regulates strain acid tolerance, yet its impact on milk fermentation remains unclear. This study aimed to elucidate the mechanism of luxS and pfs gene overexpression in the luxS/AI-2 system of S. thermophilus ABT-T on fermented milk quality using metabolomics. Results showed that pfs gene overexpression had a greater impact on milk quality than the wild-type strain or luxS gene overexpression strain. Overexpression of the pfs gene significantly enhanced AI-2 secretion, reducing fermented milk pH, increasing acidity, improving fermented milk protein hydrolysis, and altering texture and water-holding capacity. Nineteen volatile flavor compounds were identified, with decreased ketone compounds due to the pfs gene overexpression. KEGG analysis suggested significant alterations in amino acid metabolism pathways due to the pfs gene overexpression. This study provides insights into the role of QS in fermented foods.

Molecular traits of MAPK kinases and the regulatory mechanism of GhMAPKK5 alleviating drought/salt stress in cotton.

Mitogen-activated protein kinase kinases (MAPKKs) play a critical role in the mitogen-activated protein kinase (MAPK) signaling pathway, transducing external stimuli into intracellular responses and enabling plant adaptation to environmental challenges. Most research has focused on the model plant Arabidopsis (Arabidopsis thaliana). The systematic analysis and characterization of MAPKK genes across different plant species, particularly in cotton (Gossypium hirsutum), are somewhat limited. Here, we identified MAPKK family members from 66 different species, which clustered into 5 different sub-groups, and MAPKKs from four cotton species clustered together. Through further bioinformatic and expression analysis, GhMAPKK5 was identified as the most responsive MAPKK member to salt and drought stress among the 23 MAPKKs identified in Gossypium hirsutum. Silencing GhMAPKK5 in cotton through virus-induced gene silencing (VIGS) led to quicker wilting under salt and drought conditions, while overexpressing GhMAPKK5 in Arabidopsis enhanced root growth and seed germination under these stresses, demonstrating GhMAPKK5's positive role in stress tolerance. Transcriptomics and Yeast-Two-Hybrid assays revealed a MAPK cascade signal module comprising GhMEKK (Mitogen-activated protein kinase kinase kinases)3/8/31-GhMAPKK5-GhMAPK11/23. This signaling cascade may play a role in managing drought and salt stress by regulating transcription factor genes, such as WRKYs, which are involved in the biosynthesis and transport pathways of ABA, proline, and RALF. This study is highly important for further understanding the regulatory mechanism of MAPKK in cotton, contributing to its stress tolerance and offering potential in targets for genetic enhancement.

Receptor-like cytoplasmic kinase 58 reduces tolerance of maize seedlings to low magnesium via promoting H2O2 over-accumulation.

KEY MESSAGE: ZmRLCK58, a negative growth regulator, reduces tolerance of maize seedlings to low Mg via enhancing H2O2 accumulation in the shoot. Magnesium (Mg) deficiency is one of critical limiting factors for crop production in widespread acidic soils worldwide. However, the molecular mechanism of crop response to Mg deficiency is still largely unclear. Here, we found higher concentrations of H2O2, soluble sugars, and starch (1.5-, 1.9-, and 1.4-fold, respectively) in the shoot of low-Mg-treated maize seedlings, compared with Mg sufficient plants under hydroponic culture. Consistent with over-accumulation of H2O2, transcriptome profiling revealed significant enrichment of 175 differentially expressed genes (DEGs) in "response to oxygen-containing compound" out of 641 DEGs in the shoot under low Mg. Among 175 DEGs, a down-regulated receptor-like cytoplasmic kinase ZmRLCK58 underwent a recent duplication event before Poaceae divergence and was highly expressed in the maize shoot. ZmRLCK58 overexpression enhanced H2O2 accumulation in shoots by 21.3% and 29.8% under control and low-Mg conditions, respectively, while reducing biomass accumulation compared with wild-type plants. Low Mg further led to 39.7% less starch accumulation in the ZmRLCK58 overexpression shoot and lower Mg utilization efficiency. Compared with wild-type plants, overall down-regulated expression of genes related to response to carbohydrate, photosynthesis, H2O2 metabolic, oxidation-reduction, and ROS metabolic processes in ZmRLCK58 overexpression lines preconditioned aforementioned physiological alterations. Together, ZmRLCK58, as a negative growth regulator, reduces tolerance of maize seedlings to low Mg via enhancing H2O2 accumulation.

Insertion with long target duplication in polymyxin B-induced resistant mutant of Salmonella.

OBJECTIVES: A Salmonella enterica subsp. diarizonae (hereafter S. diarizonae) clinical strain S499 demonstrated unique genomic features. The strain S499 was treated with polymyxin B in vitro to investigate the mechanism of resistance. METHODS: S499 was treated with polymyxin B by increasing concentration gradually to obtain a resistant mutant S499V. Whole genomes of the two strains were sequenced using Illumina HiSeq X-10 and PacBio RS II platforms. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to compare the gene expression. RESULTS: The chromosome of strain S499 contained a 40-kb DNA region that was replicated after treatment with polymyxin B and generated a triple tandem DNA repeat region in the chromosome of mutant strain S499V. This repeat region in S499V was flanked by IS1 and contained pmrD, pmrG, and arnBCADTEF operon. In comparison to the homologous 40-kb DNA region of strain S499, a few genes in the repeat DNA region of strain S499V contained truncating mutations that generate two open reading frames (ORFs). The expression of pmrD, pmrG, and arnT was significantly upregulated in S499V. CONCLUSION: The duplication and overexpression of pmrD, pmrG, and arnT operon may be responsible for the polymyxin B resistance of mutant strain S499V.

A Sweet Potato MYB Transcription Factor IbMYB330 Enhances Tolerance to Drought and Salt Stress in Transgenic Tobacco.

MYB transcription factors (TFs) play vital roles in plant growth, development, and response to adversity. Although the MYB gene family has been studied in many plant species, there is still little known about the function of R2R3 MYB TFs in sweet potato in response to abiotic stresses. In this study, an R2R3 MYB gene, IbMYB330 was isolated from sweet potato (Ipomoea batatas). IbMYB330 was ectopically expressed in tobacco and the functional characterization was performed by overexpression in transgenic plants. The IbMYB330 protein has a 268 amino acid sequence and contains two highly conserved MYB domains. The molecular weight and isoelectric point of IbMYB330 are 29.24 kD and 9.12, respectively. The expression of IbMYB330 in sweet potato is tissue-specific, and levels in the root were significantly higher than that in the leaf and stem. It showed that the expression of IbMYB330 was strongly induced by PEG-6000, NaCl, and H2O2. Ectopic expression of IbMYB330 led to increased transcript levels of stress-related genes such as SOD, POD, APX, and P5CS. Moreover, compared to the wild-type (WT), transgenic tobacco overexpression of IbMYB330 enhanced the tolerance to drought and salt stress treatment as CAT activity, POD activity, proline content, and protein content in transgenic tobacco had increased, while MDA content had decreased. Taken together, our study demonstrated that IbMYB330 plays a role in enhancing the resistance of sweet potato to stresses. These findings lay the groundwork for future research on the R2R3-MYB genes of sweet potato and indicates that IbMYB330 may be a candidate gene for improving abiotic stress tolerance in crops.

Novel strategy of S. mutans gcrR gene over-expression plus antibacterial dimethylaminohexadecyl methacrylate suppresses biofilm acids and reduces dental caries in rats.

OBJECTIVE: Streptococcus mutans (S. mutans) is a major contributor to dental caries, with its ability to synthesize extracellular polysaccharides (EPS) and biofilms. The gcrR gene is a regulator of EPS synthesis and biofilm formation. The objectives of this study were to investigate a novel strategy of combining gcrR gene over-expression with dimethylaminohexadecyl methacrylate (DMAHDM), and to determine their in vivo efficacy in reducing caries in rats for the first time. METHODS: Two types of S. mutans were tested: Parent S. mutans; and gcrR gene over-expressed S. mutans (gcrR OE S. mutans). Bacterial minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were measured with DMAHDM and chlorhexidine (CHX). Biofilm biomass, polysaccharide, lactic acid production, live/dead staining, colony-forming units (CFUs), and metabolic activity (MTT) were evaluated. A Sprague-Dawley rat model was used with parent S. mutans and gcrR OE S. mutans colonization to determine caries-inhibition in vivo. RESULTS: Drug-susceptibility of gcrR OE S. mutans to DMAHDM or CHX was 2-fold higher than that of parent S. mutans. DMAHDM reduced biofilm CFU by 3-4 logs. Importantly, the combined gcrR OE S. mutans+ DMAHDM dual strategy reduced biofilm CFU by 5 logs. In the rat model, the parent S. mutans group had a higher cariogenicity in dentinal (Dm) and extensive dentinal (Dx) regions. The DMAHDM + gcrR OE group reduced the Dm and Dx caries to only 20 % and 0 %, those of parent S. mutans + PBS control group (p < 0.05). The total caries severity of gcrR OE + DMAHDM group was decreased to 51 % that of parent S. mutans control (p < 0.05). SIGNIFICANCE: The strategy of combining S. mutans gcrR over-expression with antibacterial monomer reducing biofilm acids by 97 %, and reduced in vivo total caries in rats by 48 %. The gcrR over-expression + DMAHDM strategy is promising for a wide range of dental applications to inhibit caries and protect tooth structures.

Analysis of chitinase gene family in barley and function study of HvChi22 involved in drought tolerance.

BACKGROUND: Chitinase (Chi) is a pathogenesis-related protein, also reported to play an important role in plant responses to abiotic stress. However, its role in response to abiotic stress in barley is still unclear. RESULTS: In this study, a total of 61 Chi gene family members were identified from the whole genome of wild barley EC_S1. Phylogenetic analysis suggested that these family genes were divided into five groups. Among these genes, four pairs of collinearity genes were discovered. Besides, abundant cis-regulatory elements, including drought response element and abscisic acid response element were identified in the promoter regions of HvChi gene family members. The expression profiles revealed that most HvChi family members were significantly up-regulated under drought stress, which was also validated by RT-qPCR measurements. To further explore the role of Chi under drought stress, HvChi22 was overexpressed in Arabidopsis. Compared to wild-type plants, overexpression of HvChi22 enhanced drought tolerance by increasing the activity of oxidative protective enzymes, which caused less MDA accumulation. CONCLUSION: Our study improved the understanding of the Chi gene family under drought stress in barley, and provided a theoretical basis for crop improvement strategies to address the challenges posed by changing environmental conditions.

Reverse Genetics Applied to Immunobiology of Tumor Necrosis Factor, a Multifunctional Cytokine.

Tumor necrosis factor (TNF) is one of many cytokines - protein molecules responsible for communication between the cells of immune system. TNF was discovered and given its grand name because of its striking antitumor effects in experimental systems, but its main physiological functions in the context of whole organism turned out to be completely unrelated to protection against tumors. This short review discusses "man-made" mouse models generated by early genome-editing technologies, which enabled us to establish true functions of TNF in health and certain diseases as well as to unravel potential strategies for improving therapy of TNF-dependent diseases.

Integration of measurable residual disease by WT1 gene expression and flow cytometry identifies pediatric patients with high risk of relapse in acute myeloid leukemia.

Background: Molecular testing plays a pivotal role in monitoring measurable residual disease (MRD) in acute myeloid leukemia (AML), aiding in the refinement of risk stratification and treatment guidance. Wilms tumor gene 1 (WT1) is frequently upregulated in pediatric AML and serves as a potential molecular marker for MRD. This study aimed to evaluate WT1 predictive value as an MRD marker and its impact on disease prognosis. Methods: Quantification of WT1 expression levels was analyzed using the standardized European Leukemia Network real-time quantitative polymerase chain reaction assay (qRT-PCR) among a cohort of 146 pediatric AML patients. Post-induction I and intensification I, MRD response by WT1 was assessed. Patients achieving a ≥2 log reduction in WT1MRD were categorized as good responders, while those failing to reach this threshold were classified as poor responders. Results: At diagnosis, WT1 overexpression was observed in 112 out of 146 (76.7%) patients. Significantly high levels were found in patients with M4- FAB subtype (p=0.018) and core binding fusion transcript (CBF) (RUNX1::RUNX1T1, p=0.018, CBFB::MYH11, p=0.016). Following induction treatment, good responders exhibited a reduced risk of relapse (2-year cumulative incidence of relapse [CIR] 7.9% vs 33.2%, p=0.008). Conversely, poor responders' post-intensification I showed significantly lower overall survival (OS) (51% vs 93.2%, p<0.001), event-free survival (EFS) (33.3% vs 82.6%, p<0.001), and higher CIR (66.6% vs 10.6%, p<0.001) at 24 months compared to good responders. Even after adjusting for potential confounders, it remained an independent adverse prognostic factor for OS (p=0.04) and EFS (p=0.008). High concordance rates between WT1-based MRD response and molecular MRD were observed in CBF patients. Furthermore, failure to achieve either a 3-log reduction by RT-PCR or a 2-log reduction by WT1 indicated a high risk of relapse. Combining MFC-based and WT1-based MRD results among the intermediate-risk group identified patients with unfavorable prognosis (positive predictive value [PPV] 100%, negative predictive value [NPV] 85%, and accuracy 87.5%). Conclusion: WT1MRD response post-intensification I serves as an independent prognostic factor for survival in pediatric AML. Integration of WT1 and MFC-based MRD results enhances the reliability of MRD-based prognostic stratification, particularly in patients lacking specific leukemic markers, thereby influencing treatment strategies.

BrTTG1 regulates seed coat proanthocyanidin formation through a direct interaction with structural gene promoters of flavonoid pathway and glutathione S-transferases in Brassica rapa L.

Introduction: Seed coat color is a significant agronomic trait in horticultural crops such as Brassica rapa which is characterized by brown or yellow seed coat coloration. Previous Brassica rapa studies have shown that BrTTG1 is responsible for seed coat proanthocyanidin formation, which is dependent on the MYB-bHLH-WD40 complex, whereas some studies have reported that TRANSPARENT TESTA GLABRA 1 (TTG1) directly interacts with the structural gene promoters of the flavonoid pathway. Methods: Herein, the brown-seeded inbred B147 and ttg1 yellow-seeded inbred B80 mutants were used as plant materials for gene expression level analysis, gene promoter clone and transient overexpression. Results: The analysis identified eleven structural genes involved in the flavonoid biosynthesis pathway, which are potentially responsible for BrTTG1- dependent seed coat proanthocyanidin formation. The promoters of these genes were cloned and cis-acting elements were identified. Yeast one-hybrid and dual-luciferase assays confirmed that BrTTG1 directly and independently interacted with proCHS-Bra008792, proDFR-Bra027457, proTT12-Bra003361, proTT19-Bra008570, proTT19-Bra023602 and proAHA10-Bra016610. A TTG1-binding motif (RTWWGTRGM) was also identified. Overexpression of TTG1 in the yellow-seed B. rapa inbred induced proanthocyanidin accumulation by increasing the expression levels of related genes. Discussion: Our study unveiled, for the first time, the direct interaction between TTG1 and the promoters of the flavonoid biosynthesis pathway structural genes and glutathione S-transferases in Brassica rapa. Additionally, we have identified a novel TTG1-binding motif, providing a basis for further exploration into the function of TTG1 and the accumulation of proanthocyanidins in seed coats.

Coupling of gene regulation and carrier modification manipulates bacterial biofilms as robust living catalysts.

The biofilm of an engineered strain is limited by slow growth and low yield, resulting in an unsatisfactory ability to resist external stress and promote catalytic efficiency. Here, biofilms used as robust living catalysts were manipulated through dual functionalized gene regulation and carrier modification strategies. The results showed that gene overexpression regulates the autoinducer-2 activity, extracellular polymeric substance content and colony behavior of Escherichia coli, and the biofilm yield of csgD overexpressed strains increased by 79.35 % compared to that of the wild type strains (p < 0.05). In addition, the hydrophilicity of polyurethane fibres modified with potassium dichromate increased significantly, and biofilm adhesion increased by 105.80 %. Finally, the isoquercitrin yield in the catalytic reaction of the biofilm reinforced by the csgD overexpression strain and the modified carrier was 247.85 % higher than that of the untreated group. Overall, this study has developed engineered strains biofilm with special functions, providing possibilities for catalytic applications.

CRISPR Activation in Mouse Trophoblast Stem Cells.

The placenta is a vital organ that regulates nutrient supply to the developing embryo during gestation. In mice, the placenta is composed of trophoblast lineage and mesodermal derivatives, which merge through the chorioallantoic fusion process in a critical event for the progression of placenta development. The trophoblast lineage is derived from self-renewing, multipotent cells known as mouse trophoblast stem cells (mTSCs). These cells are a valuable tool that allows scientists to comprehend the signals regulating major placental cell types' self-renewal and differentiation capacity. Recent advances in CRISPR-Cas9 genome editing applied in mTSCs have provided novel insights into the molecular networks involved in placentation. Here, we present a comprehensive CRISPR activation (CRISPRa) protocol based on the CRISPR/gRNA-directed synergistic activation mediator (SAM) method to overexpress specific target genes in mTSCs.

The cold-responsive C-repeat binding factors in Betula platyphylla Suk. positively regulate cold tolerance.

Cold stress is one of the most destructive abiotic stresses limiting plant growth and development. CBF (C-repeat binding factor) transcription factors and their roles in cold response have been identified in Arabidopsis as well as several other plant species. However, the biological functions and related molecular mechanisms of CBFs in birch (Betula platyphylla Suk.) remain undetermined. In this study, five cold-responsive BpCBF genes, BpCBF1, BpCBF2, BpCBF7, BpCBF10 and BpCBF12 were cloned. Via protoplast transformation, BpCBF7 was found to be localized in nucleus. The result of yeast one hybrid assay validated the binding of BpCBF7 to the CRT/DRE (C-repeat/dehydration responsive element) elements in the promoter of BpERF1.1 gene. By overexpressing and repressing BpCBFs in birch plants, it was proven that BpCBFs play positive roles in the cold tolerance. At the metabolic level, BpCBFs OE lines had lower ROS accumulation, as well as higher activities of antioxidant enzymes (SOD, POD and CAT) and higher accumulation of protective substances (soluble sugar, soluble protein and proline). Via yeast one hybrid and co-transformation of effector and reporter vectors assay, it was proven that BpCBF7 can regulate the expression of BpERF5 and BpZAT10 genes by directly binding to their promoters. An interacting protein of BpCBF7, BpWRKY17, was identified by yeast two hybrid library sequencing and the interaction was validated with in vivo methods. These results indicates that BpCBFs can increase the cold tolerance of birch plants, partly by gene regulation and protein interaction. This study provides a reference for the research on CBF transcription factors and genetic improvement of forest trees upon abiotic stresses.

New Advances in the Study of Regulation of Tomato Flowering-Related Genes Using Biotechnological Approaches.

The tomato is a convenient object for studying reproductive processes, which has become a classic. Such complex processes as flowering and fruit setting require an understanding of the fundamental principles of molecular interaction, the structures of genes and proteins, the construction of signaling pathways for transcription regulation, including the synchronous actions of cis-regulatory elements (promoter and enhancer), trans-regulatory elements (transcription factors and regulatory RNAs), and transposable elements and epigenetic regulators (DNA methylation and acetylation, chromatin structure). Here, we discuss the current state of research on tomatoes (2017-2023) devoted to studying the function of genes that regulate flowering and signal regulation systems using genome-editing technologies, RNA interference gene silencing, and gene overexpression, including heterologous expression. Although the central candidate genes for these regulatory components have been identified, a complete picture of their relationship has yet to be formed. Therefore, this review summarizes the latest achievements related to studying the processes of flowering and fruit set. This work attempts to display the gene interaction scheme to better understand the events under consideration.

A toolbox for genetic manipulation in intestinal Clostridium symbiosum.

Gut microbes are closely related with human health, but remain much to learn. Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors. However, the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies. Here, we obtained the complete genome sequence of C. symbiosum ATCC 14940, a representative strain of C. symbiosum. On this basis, we further developed a series of genetic manipulation methods for this bacterium. Firstly, following the identification of a functional replicon pBP1 in C. symbiosum ATCC 14940, a highly efficient conjugative DNA transfer method was established, enabling the rapid introduction of exogenous plasmids into cells. Next, we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium, reaching over 60 % repression for most of the chosen genes as well as efficient deletion (>90 %) of three target genes. Finally, this toolbox was used for the identification of crucial functional genes, involving growth, synthesis of important metabolites, and virulence of C. symbiosum ATCC 14940. Our work has effectively established and optimized genome editing methods in intestinal C. symbiosum, thereby providing strong support for further basic and application research in this bacterium.

Overexpression of BAPT and DBTNBT genes in Taxus baccata in vitro cultures to enhance the biotechnological production of paclitaxel.

Paclitaxel is one of the most effective anticancer drugs ever developed. Although the most sustainable approach to its production is provided by plant cell cultures, the yield is limited by bottleneck enzymes in the taxane biosynthetic pathway: baccatin-aminophenylpropanoyl-13-O-transferase (BAPT) and 3'-N-debenzoyltaxol N-benzoyltransferase (DBTNBT). With the aim of enhancing paclitaxel production by overcoming this bottleneck, we obtained distinct lines of Taxus baccata in vitro roots, each independently overexpressing either of the two flux-limiting genes, BAPT or DBTNBT, through a Rhizobium rhizogenes A4-mediated transformation. Due to the slow growth rate of the transgenic Taxus roots, they were dedifferentiated to obtain callus lines and establish cell suspensions. The transgenic cells were cultured in a two-stage system and stimulated for taxane production by a dual elicitation treatment with 1 µm coronatine plus 50 mm of randomly methylated-ß-cyclodextrins. A high overexpression of BAPT (59.72-fold higher at 48 h) and DBTNBT (61.93-fold higher at 72 h) genes was observed in the transgenic cell cultures, as well as an improved taxane production. Compared to the wild type line (71.01 mg/L), the DBTNBT line produced more than four times higher amounts of paclitaxel (310 mg/L), while the content of this taxane was almost doubled in the BAPT line (135 mg/L). A transcriptional profiling of taxane biosynthetic genes revealed that GGPPS, TXS and DBAT genes were the most reactive to DBTNBT overexpression and the dual elicitation, their expression increasing gradually and constantly. The same genes exhibited a pattern of isolated peaks of expression in the elicited BAPT-overexpressing line.

ALS gene overexpression and enhanced metabolism conferring Digitaria sanguinalis resistance to nicosulfuron in China.

Crabgrass (Digitaria sanguinalis) is a common malignant weed in corn fields in China. Recently, the acetolactate synthase (ALS) inhibitor, nicosulfuron, has shown decreasing efficacy against crabgrass. In order to elucidate the molecular basis of resistance to nicosulfuron in crabgrass, we conducted bioassays, combined with gene sequence analysis, relative expression and relative copy number analysis, to characterize resistance in crabgrass populations collected from Beijing, Heilongjiang, Jilin and Anhui provinces. Whole-plant dose-response results indicated that only population collected in Heilongjiang province (HLJ) had developed low level of resistance to nicosulfuron compared with the sensitive population (SD22). No known resistant mutation of ALS gene was found in HLJ population. The real-time fluorescence quantitative PCR results showed that the ALS gene copy number did not differ significantly between the HLJ and SD22 populations. However, the ALS gene expression in the HLJ was 2.07-fold higher than that of the SD22 population at 24 h after treatment with nicosulfuron. Pretreatment with the cytochrome P450 (CYP450) inhibitor malathion, piperonyl butoxide (PBO), and the glutathione S-transferase (GST) inhibitor 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) all partially reversed HLJ resistance. Among them, the synergistic effect of PBO and nicosulfuron is the most significant. This is the first report of resistance to nicosulfuron in crabgrass through ALS gene overexpression and possible metabolic resistance.

Multiplex metabolic pathway engineering of Monascus pilosus enhances lovastatin production.

Monascus sp. is an important food microbial resource with the production of cholesterol-lowering agent lovastatin and other healthy metabolites. However, the mycotoxin citrinin naturally produced by Monascus sp. and the insufficient productivity of lovastatin limit its large-scale use in food industry. The aim of this paper is to modify a lovastatin-producing strain Monascus pilosus GN-01 through metabolic engineering to obtain a citrinin-free M. pilosus strain with higher yield of lovastatin. The citrinin synthesis regulator gene ctnR was firstly disrupted to obtain GN-02 without citrinin production. Based on that, the lovastatin biosynthesis genes (mokC, mokD, mokE, mokF, mokH, mokI, and LaeA) were, respectively, overexpressed, and pigment-regulatory gene (pigR) was knocked out to improve lovastatin production. The results indicated ctnR inactivation effectively disrupted the citrinin release by M. pilosus GN-01. The overexpression of lovastatin biosynthesis genes and pigR knockout could lead higher contents of lovastatin, of which pigR knockout strain achieved 76.60% increase in the yield of lovastatin compared to GN-02. These studies suggest that such multiplex metabolic pathway engineering in M. pilosus GN-01 is promising for high lovastatin production by a safe strain for application in Monascus-related food. KEY POINTS: • Disruption of the regulator gene ctnR inhibited citrinin production of M. pilosus. • Synchronous overexpression of biosynthesis gene enhanced lovastatin production. • pigR knockout enhanced lovastatin of ΔctnR strain of M. pilosus.