Production of 2,3-butanediol from glucose and cassava hydrolysates by metabolically engineered industrial polyploid Saccharomyces cerevisiae

Background 2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol. Results Endogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h. Conclusions The microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.This study is funded by National Research Foundation of Korea (2011-0031359)

Compressed sensing fMRI using gradient-recalled echo and EPI sequences

Compressed sensing (CS) may be useful for accelerating data acquisitions in high-resolution fMRI. However, due to the inherent slow temporal dynamics of the hemodynamic signals and concerns of potential statistical power loss, the CS approach for fMRI (CS-fMRI) has not been extensively investigated. To evaluate the utility of CS in fMRI application, we systematically investigated the properties of CS-fMRI using computer simulations and in vivo experiments of rat forepaw sensory and odor stimulations with gradient-recalled echo (GRE) and echo planar imaging (EPI) sequences. Various undersampling patterns along the phase-encoding direction were studied and k-t FOCUSS was used as the CS reconstruction algorithm, which exploits the temporal redundancy of images. Functional sensitivity, specificity, and time courses were compared between fully-sampled and CS-fMRI with reduction factors of 2 and 4. CS-fMRI with GRE, but not with EPI, improves the statistical sensitivity for activation detection over the fully sampled data when the ratio of the fMRI signal change to noise is low. CS improves the temporal resolution and temporal noise correlations. While CS reduces the functional response amplitudes, the noise variance is also reduced to make the overall activation detection more sensitive. Consequently, CS is a valuable fMRI acceleration approach, especially for GRE fMRI studies

A new marine ciliate, Metaurostylopsis antarctica nov. spec. (Ciliophora, Urostylida) from the Antarctic Ocean

In this study, a new marine urostylid ciliate, Metaurostylopsis antarctica nov. spec. collected from the Antarctic Ocean was investigated using morphological, morphometrical, and molecular methods. Metaurostylopsis antarctica nov. spec. is characterized as follows: slender to ellipsoid form in body shape; two types of cortical granules, ellipsoid large one (type I, yellow-green, 1.5 × 1 μm) in rows along dorsal kineties and cirri, circular small one (type II, colourless, 0.3 μm in diameter) scattered throughout whole body; 19–24 adoral membranelles, 4 frontal cirri, 2–5 frontoterminal cirri, 1 buccal and 2 transverse cirri; 3–5 midventral pairs, 10–15 cirri of midventral row; 1 right and 2 left marginal rows; 3 dorsal kineties; about 43 macronuclear nodules. This new species mainly differs from the congeners by the number of marginal rows (1 vs. 3 or more on right side; 2 vs. 3 or more on left side). In addition, proter’s oral primordium  developed on the right side of the oral cavity (vs. in center of oral cavity), and the rightmost anlage splits into two parts, nam ely, the frontoterminal cirri and a transverse cirrus (vs. only frontoterminal cirri). Inter-specific dissimilarities of the SSU rRNA gene between the congeners range from 3.3 to 4.4%

Seasonal variations in carbon, nitrogen and phosphorus concentrations and C:N:P stoichiometry in different organs of a Larix principis-rupprechtii Mayr. plantation in the Qinling Mountains, China

Understanding how concentrations of elements and their stoichiometry change with plant growth and age is critical for predicting plant community responses to environmental change. Weusedlong-term field experiments to explore how the leaf, stem and root carbon (C), nitrogen (N) and phosphorous (P) concentrations and their stoichiometry changed with growth and stand age in a L.principis-rupprechtii Mayr. plantation from 2012–2015 in the Qinling Mountains, China. Our results showed that the C, N and P concentrations and stoichiometric ratios in different tissues of larch stands were affected by stand age, organ type andsampling month and displayed multiple correlations with increased stand age in different growing seasons. Generally, leaf C and N concentrations were greatest in the fast-growing season, but leaf P concentrations were greatest in the early growing season. However, no clear seasonal tendencies in the stem and root C, N and P concentrations were observed with growth. In contrast to N and P, few differences were found in organ-specific C concentrations. Leaf N:P was greatest in the fast-growing season, while C:N and C:P were greatest in the late-growing season. No clear variations were observed in stem and root C:N, C:P andN:Pthroughout the entire growing season, but leaf N:P was less than 14, suggesting that the growth of larch stands was limited by N in our study region. Compared to global plant element concentrations and stoichiometry, the leaves of larch stands had higher C, P, C:NandC:PbutlowerNandN:P,andtherootshadgreater PandC:NbutlowerN,C:Pand N:P. Our study provides baseline information for describing the changes in nutritional elements with plant growth, which will facilitates plantation forest management and restoration, and makes avaluable contribution to the global data pool on leaf nutrition and stoichiometry

A childhood case of spinal tuberculosis misdiagnosed as muscular dystrophy

Tuberculosis is primarily a pulmonary disease, but extra-pulmonary manifestations are not uncommon, especially in children and adolescents. Ten percent of extra pulmonary tuberculosis localizes to the bones and joints, and 56% of such cases affect the spine. We treated a childhood case of spinal tuberculosis misdiagnosed as muscular dystrophy in a patient without specific constitutional symptoms. We report this case because the patient had an unusual presentation of spinal tuberculosis

Effects of the field modulation on the Hofstadter's spectrum

We study the effect of spatially modulated magnetic fields on the energy spectrum of a two-dimensional (2D) Bloch electron. Taking into account four kinds of modulated fields and using the method of direct diagonalization of the Hamiltonian matrix, we calculate energy spectra with varying system parameters (i.e., the kind of the modulation, the relative strength of the modulated field to the uniform background field, and the period of the modulation) to elucidate that the energy band structure sensitively depends on such parameters: Inclusion of spatially modulated fields into a uniform field leads occurrence of gap opening, gap closing, band crossing, and band broadening, resulting distinctive energy band structure from the Hofstadter's spectrum. We also discuss the effect of the field modulation on the symmetries appeared in the Hofstadter's spectrum in detail.Comment: 7 pages (in two-column), 10 figures (including 2 tables

Theory of disk accretion onto supermassive black holes

Accretion onto supermassive black holes produces both the dramatic phenomena associated with active galactic nuclei and the underwhelming displays seen in the Galactic Center and most other nearby galaxies. I review selected aspects of the current theoretical understanding of black hole accretion, emphasizing the role of magnetohydrodynamic turbulence and gravitational instabilities in driving the actual accretion and the importance of the efficacy of cooling in determining the structure and observational appearance of the accretion flow. Ongoing investigations into the dynamics of the plunging region, the origin of variability in the accretion process, and the evolution of warped, twisted, or eccentric disks are summarized.Comment: Mostly introductory review, to appear in "Supermassive black holes in the distant Universe", ed. A.J. Barger, Kluwer Academic Publishers, in pres

A Prospective Randomized Trial of Either Famotidine or Pantoprazole for the Prevention of Bleeding after Endoscopic Submucosal Dissection

Endoscopic submucosal dissection (ESD) has been reported to have a higher bleeding rate than conventional methods. However, there are few reports on whether a proton pump inhibitor or a histamine2-receptor antagonist is the more effective treatment for preventing bleeding after ESD. In a prospective trial, patients undergoing ESD due to gastric adenoma or adenocarcinoma were randomly assigned to pantoprazole or famotidine. Both drugs were given intravenously for the first 2 days, thereafter by mouth. Eighty-five in the pantoprazole group and 79 in the famotidine group were included for analysis. Primary outcome measure was the delayed bleeding rate. Clinical characteristics were not different between the two groups. The delayed bleeding rate was significantly lower in the pantoprazole group compared with the famotidine group (3.5% vs. 12.7%, p=0.031). On multivariate analysis, the preventive use of pantoprazole (relative hazard: 0.220, 95% CI: 0.051- 0.827, p=0.026) and the specimen size (≥34 mm, relative hazard: 4.178, 95% CI: 1.229-14.197, p=0.022) were two independent factors predictive of delayed bleeding. There were no significant differences in en bloc and complete resection rate between the two groups. In conclusion, pantoprazole is more effective than famotidine for the prevention of delayed bleeding after ESD

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including safe harboring techniques shown in other organisms.