Engineering of Corynebacterium glutamicum for the secretory production of recombinant proteins via Tat-dependent pathway

Corynebacterium glutamicum, which has been an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins since it exhibits numerous ideal features for protein secretion: (i) it has a single cellular membrane as a gram-positive bacterium, which allows proteins to be easily secreted into the extracellular medium. (ii) C. glutamicum secretes only a few endogenous proteins into the culture medium, which allows the simpler purification of target proteins in downstream process. (iii), secreted proteins from C. glutamicum can be kept stable because extracellular protease activity is rarely detectable. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. To achieve this goal first, we engineered several genetic parts in C. glutamicum: (i) synthetic promoters, (ii) plasmid copy number, (iii) signal peptides, (iv) co-expression of secretion machinery proteins. Using the engineered host-vector systems, gram-scale production of recombinant proteins could be achieved in fed-batch cultivation

Engineering of Klebsiella oxytoca capable of simultaneous utilization of multiple sugars for the production of 2, 3- Butanediol

From various biomasses such as lignocellulose and microalgae, many kinds of monosaccharides including glucose, galactose, xylose, mannose, ribose, rhamnose, and fucose can be obtained. Among them, galactose and xylose are the major carbon sources except for glucose in nature, and both sugars can serve as additive for the production of desired chemicals in the glucose-based fermentation. However, in many microorganisms, the glucose hampers utilization of galactose and xylose until depletion of glucose owing to Carbon Catabolite Repression (CCR) mechanism, which has been a big hurdle for the development of bioprocess utilizing multiple carbon sugars. Here, we developed Klebsiella oxytoca capable of simultaneous utilization of three sugars including glucose, galactose and xylose for the fermentative production of 2,3-butanediol which is a vital platform compound, used as liquid fuel and chemical raw material. To eliminate CCR and utilize multiple sugars, the phosphotransferase system (PTS) which is the main transporter for glucose was disrupted, in which cells could uptake glucose through alternative pathway and the transport system for other sugars could be activated. To verify the removal of CCR by disruption of PTS, the engineered strain was cultivated with two or three sugars and, we found that the simultaneous consumption of galactose and xylose was achieved although glucose consumption rate was decreased a little. At the time point of complete consumption of glucose, most galactose was also consumed and, about 30 % of xylose was consumed before glucose depletion. Under the simultaneous utilization of galactose and xylose along with glucose, 2,3-butaneidol was also successfully produced as high as 0.3 g/g, which yield is similar as that in cultivation with glucose as a sole carbon source. To the best of our knowledge, this is the first example of CCR elimination in K. oxytoca and, we think that our strategy sheds new light on an engineering of K. oxytoca for commercial exploitation of biomass to produce value-added products

Activation of MAPK and CREB by GM1 Induces Survival of RGCs in the Retina with Axotomized Nerve

PURPOSE. Neuronal cells undergo apoptosis when the supply of neurotrophic factor is limited by injury, trauma, or neurodegenerative disease. Ganglioside has both neuritogenic and neurotropic functions. Exogenously administered monosialoganglioside (GM1) has been shown to have a stimulatory effect on neurite outgrowth and to prevent degeneration of neuronal cells in the central nervous system. Even though GM1 has been shown to mimic, or have synergy with, neurotrophic factors, the neuroprotective mechanism of GM1 has not been well understood. In this study, optic nerve transection, or axotomy, was used as an in vivo model system for injury, to examine the protective mechanism of GM1 in injured retinal ganglion cells. METHODS. GM1 was injected into the vitreous body before axotomy, and the protective effect of GM1 observed with regard to activation of mitogen-activated protein kinase (MAPK) and phosphorylation of cAMP-responsive elementbinding (CREB) protein. Activation of MAPK and CREB were examined by Western blot analysis and immunohistochemistry, and the surviving retinal ganglion cells were counted after retrograde fluorescence labeling. RESULTS. GM1 inhibited the degeneration of axotomized retinal ganglion cells. In addition, GM1 enhanced the activation of MAPK and CREB with the treatment of GM1 in the retina with axotomized nerve. Treatment of MAPK inhibitor PD98059 with GM1 reduced the protective action of GM1 and prevented GM1-induced phosphorylation of CREB. CONCLUSIONS. GM1 protected the axotomized retinal ganglion cells (RGCs) from cell death after axotomy through the activation of MAPK and CREB. (Invest Ophthalmol Vis Sci

Does robot-assisted laparoscopic radical prostatectomy enable to obtain adequate oncological and functional outcomes during the learning curve? From the Korean experience

To estimate the short-term results of robot-assisted laparoscopic radical prostatectomy (RALRP) during the learning curve, in terms of surgical, oncological and functional outcomes, we conducted a prospective survey on RALRP. From July 2007, a single surgeon performed 63 robotic prostatectomies using the same operative technique. Perioperative data, including pathological and early functional results of the patient, were collected prospectively and analyzed. Along with the accumulation of the cases, the total operative time, setup time, console time and blood loss were significantly decreased. No major complication was present in any patient. Transfusion was needed in six patients; all of them were within the initial 15 cases. The positive surgical margin rate was 9.8% (5/51) in pT2 disease. The most frequent location of positive margin in this stage was the lateral aspect (60%), but in pT3 disease multiple margins were the most frequent (41.7%). Overall, 53 (84.1%) patients had totally continent status and the median time to continence was 6.56 weeks. Among 17 patients who maintained preoperative sexual activity ( Sexual Health Inventory for Men \u3e = 17), stage below pT2, followed up for \u3e 6 months with minimally one side of neurovascular bundle preservation procedure, 12 (70.6%) were capable of intercourse postoperatively, and the mean time for sexual intercourse after operation was 5.7 months. In this series, robotic prostatectomy was a feasible and reproducible technique, with a short learning curve and low perioperative complication rate. Even during the initial phase of the learning curve, satisfactory results were obtained with regard to functional and oncological outcome

Mind bomb 1 in the lymphopoietic niches is essential for T and marginal zone B cell development

Notch signaling regulates lineage decisions at multiple stages of lymphocyte development, and Notch activation requires the endocytosis of Notch ligands in the signal-sending cells. Four E3 ubiquitin ligases, Mind bomb (Mib) 1, Mib2, Neuralized (Neur) 1, and Neur2, regulate the Notch ligands to activate Notch signaling, but their roles in lymphocyte development have not been defined. We show that Mib1 regulates T and marginal zone B (MZB) cell development in the lymphopoietic niches. Inactivation of the Mib1 gene, but not the other E3 ligases, Mib2, Neur1, and Neur2, abrogated T and MZB cell development. Reciprocal bone marrow (BM) transplantation experiments revealed that Mib1 in the thymic and splenic niches is essential for T and MZB cell development. Interestingly, when BM cells from transgenic Notch reporter mice were transplanted into Mib1-null mice, the Notch signaling was abolished in the double-negative thymocytes. In addition, the endocytosis of Dll1 was impaired in the Mib1-null microenvironment. Moreover, the block in T cell development and the failure of Dll1 endocytosis were also observed in coculture system by Mib1 knockdown. Our study reveals that Mib1 is the essential E3 ligase in T and MZB cell development, through the regulation of Notch ligands in the thymic and splenic microenvironments