A noninvasive and selective therapy, photodynamic therapy (PDT) is widely researched in clinical fields; however, the lower efficiency of PDT can induce unexpected side effects. Mitochondria are extensively researched as target sites to maximize PDT effects because they play crucial roles in metabolism and can be used as cancer markers due to their high transmembrane potential. Here, a mitochondria targeting photodynamic therapeutic agent (MitDt) is developed. This photosensitizer is synthesized from heptamethine cyanine dyes, which are conjugated or modified as follows. The heptamethine meso‐position is conjugated with a triphenylphosphonium derivative for mitochondrial targeting, the N‐alkyl side chain is modified for regulation of charge balance and solubility, and the indolenine groups are brominated to enhance reactive oxygen species generation (ROS) after laser irradiation. The synthesized MitDt increases the cancer uptake efficiency due to the lipo‐cationic properties of the triphenylphosphonium, and the PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS, the response to which triggers an apoptotic anticancer effect. Consequently, these hypotheses are demonstrated by in vitro and in vivo studies, and the results indicate strong potential for use of MitDts as efficient single‐molecule‐based PDT agents for cancer treatment.

BackgroundPichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.ResultsA fully compartmentalized metabolic model of P. pastoris (iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.ConclusionThe genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

Muscle development and lipid accumulation in muscle critically affect meat quality of livestock. However, the genetic factors underlying myofiber-type specification and intramuscular fat (IMF) accumulation remain to be elucidated. Using two independent intercrosses between Western commercial breeds and Korean native pigs (KNPs) and a joint linkage-linkage disequilibrium analysis, we identified a 488.1-kb region on porcine chromosome 12 that affects both reddish meat color (a*) and IMF. In this critical region, only the MYH3 gene, encoding myosin heavy chain 3, was found to be preferentially overexpressed in the skeletal muscle of KNPs. Subsequently, MYH3-transgenic mice demonstrated that this gene controls both myofiber-type specification and adipogenesis in skeletal muscle. We discovered a structural variant in the promotor/regulatory region of MYH3 for which Q allele carriers exhibited significantly higher values of a* and IMF than q allele carriers. Furthermore, chromatin immunoprecipitation and cotransfection assays showed that the structural variant in the 5′-flanking region of MYH3 abrogated the binding of the myogenic regulatory factors (MYF5, MYOD, MYOG, and MRF4). The allele distribution of MYH3 among pig populations worldwide indicated that the MYH3 Q allele is of Asian origin and likely predates domestication. In conclusion, we identified a functional regulatory sequence variant in porcine MYH3 that provides novel insights into the genetic basis of the regulation of myofiber type ratios and associated changes in IMF in pigs. The MYH3 variant can play an important role in improving pork quality in current breeding programs.

Acetic acid is an abundant material that can be used as a carbon source by microorganisms. Despite its abundance, its toxicity and low energy content make it hard to utilize as a sole carbon source for biochemical production. To increase acetate utilization and isobutanol production with engineered Escherichia coli, the feasibility of utilizing acetate and metabolic engineering was investigated. The expression of acs, pckA, and maeB increased isobutanol production by up to 26%, and the addition of TCA cycle intermediates indicated that the intermediates can enhance isobutanol production. For isobutanol production from acetate, acetate uptake rates and the NADPH pool were not limiting factors compared to glucose as a carbon source. This work represents the first approach to produce isobutanol from acetate with pyruvate flux optimization to extend the applicability of acetate. This technique suggests a strategy for biochemical production utilizing acetate as the sole carbon source.

Recently, the bio-production of α,ω-dicarboxylic acids (DCAs) has gained significant attention, which potentially leads to the replacement of the conventional petroleum-based products. In this regard, the lipid accumulating yeast Candida tropicalis, has been recognized as a promising microbial host for DCA biosynthesis: it possess the unique ω-oxidation pathway where the terminal carbon of α-fatty acids is oxidized to form DCAs with varying chain lengths. However, despite such industrial importance, its cellular physiology and lipid accumulation capability remain largely uncharacterized. Thus, it is imperative to better understand the metabolic behavior of this lipogenic yeast, which could be achieved by a systems biological approach. To this end, herein, we reconstructed the genome-scale metabolic model of C. tropicalis, iCT646, accounting for 646 unique genes, 945 metabolic reactions, and 712 metabolites. Initially, the comparative network analysis of iCT646 with other yeasts revealed several distinctive metabolic reactions, mainly within the amino acid and lipid metabolism including the ω-oxidation pathway. Constraints-based flux analysis was, then, employed to predict the in silico growth rates of C. tropicalis which are highly consistent with the cellular phenotype observed in glucose and xylose minimal media chemostat cultures. Subsequently, the lipid accumulation capability of C. tropicalis was explored in comparison with Saccharomyces cerevisiae, indicating that the formation of "citrate pyruvate cycle" is essential to the lipid accumulation in oleaginous yeasts. The in silico flux analysis also highlighted the enhanced ability of pentose phosphate pathway as NADPH source rather than malic enzyme during lipogenesis. Finally, iCT646 was successfully utilized to highlight the key directions of C. tropicalis strain design for the whole cell biotransformation application to produce long-chain DCAs from alkanes. Biotechnol. Bioeng. 2016;113: 1993-2004. © 2016 Wiley Periodicals, Inc.

In general, high broth viscosity is a key factor to be considered in a submerged fermentation of filamentous fungi. High broth viscosity was also observed in a batch fermentation of Monascus sp. J101 at 30 degrees C. In a batch culture at 30 degrees C, most cell growth was accomplished within 48 h, which induced highly entangled clumps. The resultant high viscosity induced heterogeneity inside the fermentor, poor oxygen transfer, and low pigment yield. However, these problems could be overcome by reducing fungal growth rate through culture at low temperature (25 degrees C). Cell growth was moderate and continued for 120 h, and low viscosity was maintained. The DO levels remained at 50% or higher with good mixing. As a result, the pigment yield at 25 degrees C was 10 times greater than at 30 degrees C.

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Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neuron disease. The etiology and pathogenic mechanisms of the disease remain unknown, and there is no effective treatment. Here we show that intrathecal transplantation of human motor neurons derived from neural stem cells (NSCs) in spinal cord of the SOD1G93A mouse ALS model delayed disease onset and extended life span of the animals. When HB1.F3.Olig2 (F3.Olig2) cells, stable immortalized human NSCs encoding the human Olig2 gene, were treated with sonic hedgehog (Shh) protein for 5–7 days, the cells expressed motor neuron cell type-specific phenotypes Hb9, Isl-1 and choline acetyltransferase (ChAT). These F3.Olig2-Shh human motor neurons were transplanted intrathecally in L5–L6 spinal cord of SOD1G93A mice, and at 4 weeks post-transplantation, transplanted F3.Olig2-Shh motor neurons expressing the neuronal phenotype markers NF, MAP2, Hb9, and ChAT were found in the ventral horn of the spinal cord. Onset of clinical signs in ALS mice with F3.Olig2-Shh motor neuron implants was delayed for 7 days and life span of animals was significantly extended by 20 days. Our results indicate that this treatment modality of intrathecal transplantation of human motor neurons derived from NSCs might be of value in the treatment of ALS patients without significant adverse effects.