Antisense Silencing of the creA Gene in Aspergillus nidulans

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was approximately one-half of that achieved in a null creA mutant. Unlike results for that mutant, however, growth parameters and colony morphology in the antisense transformants were not affected.     

Cloning of a Phenol Oxidase Gene from Acremonium murorum and Its Expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.     

Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

We report the expression and production of llama variable heavy-chain antibody fragments (V_HHs) by Aspergillus awamori. Fragments encoding V_HHs were cloned in a suitable Aspergillus expression vector and transformants secreting V_HH fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V_HHs were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V_HHs were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.An erratum to this article can be found at     

Production of Phosphatase by Aspergillus awamori var. kawachii in a Low Phosphate Medium

The effect of phosphate on the production of phosphatases by Aspergillus awamori var. kawachii was studied. In a high phosphate medium, little phosphatase was produced, and the phosphatase activity was predominately for beta-glycerophosphate. In a low phosphate medium, the production of phosphatase was increased and activity for glucose-6-phosphate predominated. Medium containing 1 mg of phosphorus per 100 ml was optimal, and the amount of phosphatase produced in this medium was about 200 times that produced in a high phosphate medium. By means of column chromatography on diethylaminoethyl cellulose, the phosphatase produced in the high phosphate medium was found to be eluted mainly at fraction e; the phosphatase of the low phosphate medium was separated into fractions a, b, c, and d. Thus, the phosphatase fractions produced in the low phosphate medium were different from those of the high phosphate medium. Since no specific effect on the production of esterases was observed when various phosphate esters were used as substrates, the enzymes of phosphate metabolism appear to be activated by nonspecific phosphate sources.     

Waxy基因的RNA沉默使转基因小麦种子中直链淀粉含量下降

通过RNAi策略转化小麦,以降低小麦种子中直链淀粉的含量。小麦中直链淀粉合成的关键酶是颗粒结合型淀粉合成酶（Granule—bound starch synthase l,GBSSI,即WAXY蛋白）,通过RT—PCR方法从小麦种子中分离出Waxy基因。Southern杂交分析表明,在基因组中存在3个Waxy基因。Northern杂交分析显示出在授粉后的小麦种子中检测到Waxy mRNA。利用RNA沉默策略,将Waxy编码区683bp的正向和反向片段以及150bp内含子,连接于表达载体pCAMBIA3300中玉米ubil启动子下游。以扬麦10号授粉后15d的幼胚为外植体,利用农杆菌介导的方法进行转化。通过PCR、RT-PCR和叶片离体褪绿实验鉴定出4株转基因植株。小麦胚乳I2-KI染色和直链淀粉含量测定表明这4株转基因植株直链淀粉含量明显下降。研究结果表明Waxy基因的RNA沉默使转基因小麦种子直链淀粉的含量下降。     

Disruption of mstn Gene by CRISPR/Cas9 in Large Yellow Croaker (Larimichthys crocea)

Marine Biotechnology - The large yellow croaker (Larimichthys crocea) plays an economically vital role in the marine aquaculture in China. Suffering from infection of bacteria and protozoon, effect...     

Expression of the Cellulase (FI-CMCase) Gene of Aspergillus aculeatus in Escherichia coli

FI-CMCase cDNA of Aspergillus aculeatus was expressed in Escherichia coli by using the tac promoter of E. coli. Transformants of E. coli harboring a plasmid pHEM06 containing mature form FI-CMCase cDNA produced FI-CMCase in the cytoplasm of the cells. The enzyme from E. coli cells was purified to yield 56% and it was immunological identical to that of FI-CMCase purified from A. aculeatus.     

Pectin Methylesterase Isoforms in Tomato (Lycopersicon esculentum) Tissues (Effects of Expression of a Pectin Methylesterase Antisense Gene)

We have identified two major groups of pectin methylesterase (PME, EC 3.1.1.11) isoforms in various tissues of tomatoes (Lycopersicon esculentum). These two groups exhibited differential immuno-cross-reactivity with polyclonal antibodies raised against tomato fruit PME or flax callus PME and differences in their accumulation patterns in tissues of wild-type and transgenic tomato plants expressing a PME antisense gene. The group I isoforms with isoelectric points (pls) of 8.2, 8.4, and 8.5 are specific to fruit tissue, where they are the major forms of PME activity. The group II PME isoforms, with pl values of 9 and above, are observed in both vegetative and fruit tissues. The group I isoforms cross-react with polyclonal antibodies raised to a PME isoform purified from fruit, whereas the group II isoforms cross-react with antibodies to a PME purified from flax callus. Expression of a fruit-specific PME anti-sense gene impairs accumulation of the group I PME isoforms, with no apparent effect on the accumulation of the group II PME isoforms. The absence of any noticeable effects on growth and development of transgenic plants suggests that the group I PME isoforms are not involved in plant growth and development and may play a role under special circumstances such as cell separation during fruit ripening.     

Expression of an Aspergillus niger Phytase Gene (phyA) in Saccharomyces cerevisiae

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase’s activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60°C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.     

Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

Background

The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy.

Methods/Principal Findings

The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors.

Conclusions/Significance

The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating that the essential gene yidC is a therapeutic target of the antibacterial essential oils eugenol and carvacrol in E. coli.     

Effect of a Microbial Acid Protease Inhibitor (S-PI) on the Production of Fungal Acid Protease

Cladosporium sp. No. 45–2, an acid protease-producing microorganism, was cultured in medium containing a microbial acid protease inhibitor (S–PI). By the addition of S–PI, the amount of acid protease in the culture broth showed an increase of 50~80% over those of normal culture (S–PI-free). Acid protease was purified from the S–PI-added culture filtrate, and its enzymatic and physicochemical properties were compared with those of acid protease obtained from normal culture. It was determined that the acid protease obtained from S–PI-added culture was the same as that of normal culture, but that the productivity was increased by the addition of S–PI.

The increase in acid protease productivity is assumed to be due to a change in metabolism by the addition of S–PI.     

RNA干扰抑制乙型肝炎病毒复制及基因表达研究进展

RNA干扰(RNAinterference,RNAi)是指由21～23个核苷酸组成的双链RNA(dsRNA)所引发的生物细胞内同源基因转录后沉默的现象,是生物体在进化过程中普遍存在的一种基因调控机制。目前对由乙型肝炎病毒(HBV)引起的病毒性肝炎尚无令人满意的治疗效果,而RNA干扰技术的出现为各类慢性HBV感染的治疗开辟了新的途径。本文对RNA干扰抑制HBV复制及基因表达的研究现状、存在问题及应用前景进行了综述。     

Antisense RNA Inhibition of RbcS Gene Expression Reduces Rubisco Level and Photosynthesis in the C4 Plant Flaveria bidentis

The C4 dicot Flaveria bidentis was genetically transformed with an antisense RNA construct targeted to the nuclear-encoded gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; RbcS). RbcS mRNA levels in leaves of transformants were reduced by as much as 80% compared to wild-type levels, and extractable enzyme activity was reduced by up to 85%. There was no significant effect of transformation with the gene construct on levels of other photosynthetic enzymes. Antisense transformants with reduced Rubisco activity exhibited a stunted phenotype. Rates of photosynthesis were reduced in air at high light and over a range of CO2 concentrations but were unaffected at low light. From these results we conclude that, as is the case in C3 plants, Rubisco activity is a major determinant of photosynthetic flux in C4 plants under high light intensities and air levels of CO2.     

Production of bifunctional proteins by Aspergillus awamori: llama variable heavy chain antibody fragment (V(HH)) R9 coupled to Arthromyces ramosus peroxidase (ARP)

The Arthromyces ramosus peroxidase gene (arp) was genetically fused to either the 5′- or 3′-terminal ends of the gene encoding llama variable heavy chain antibody fragment V_HH R9, resulting in the fusion expression cassettes ARP-R9 or R9-ARP. Aspergillus awamori transformants were obtained which produced up to 30 mg l⁻¹ fusion protein in the culture medium. Both fusion proteins showed peroxidase activity in an ABTS activity test. Considerable amounts of fusion protein were detected intracellularly, suggesting that the fungus encounters problems in secreting these kind of proteins. ELISA experiments showed that ARP-R9 was less able to bind its antigen, the azo-dye RR6, as compared to R9-ARP. Furthermore, in contrast to R9-ARP, ARP-R9 bound to RR6 did not show peroxidase activity anymore. These results indicate that fusion of ARP to the C-terminus of the antibody fragment V_HH R9 (R9-ARP) is the preferred orientation.     

Inhibition of Gene Expression by Homologous Double-Stranded RNA in a Drosophila melanogasterCell Culture

Specific inhibition of gene expression by exogenous homologous double-stranded RNA (dsRNA) in invertebrates and in the early development of vertebrates is termed RNA interference. Cultured cells were cotransfected with reporter plasmids and dsRNA. The inhibitory effect on reporter gene expression depended on the extent of homology between dsRNA and the target gene. RNA interference was also studied in cells cotransfected with plasmids directing synthesis of sense and antisense RNAs. Production of antisense RNA only slightly inhibited expression of the reporter gene. Simultaneous expression of both sense and antisense RNAs caused by cotransfection by corresponding plasmids did not inhibit expression of the reporter construct.