Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

Utilizing elementary mode analysis,pathway thermodynamics,and a genetic algorithm for metabolic flux determination and optimal metabolic network design

Background  

Microbial hosts offer a number of unique advantages when used as production systems for both native and heterologous small-molecules. These advantages include high selectivity and benign environmental impact; however, a principal drawback is low yield and/or productivity, which limits economic viability. Therefore a major challenge in developing a microbial production system is to maximize formation of a specific product while sustaining cell growth. Tools to rationally reconfigure microbial metabolism for these potentially conflicting objectives remain limited. Exhaustively exploring combinations of genetic modifications is both experimentally and computationally inefficient, and can become intractable when multiple gene deletions or insertions need to be considered. Alternatively, the search for desirable gene modifications may be solved heuristically as an evolutionary optimization problem. In this study, we combine a genetic algorithm and elementary mode analysis to develop an optimization framework for evolving metabolic networks with energetically favorable pathways for production of both biomass and a compound of interest.     

Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury – A Microarray Study

Recently, we have shown that inhalation of hydrogen sulfide (H₂S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H₂S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H₂S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H₂S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H₂S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H₂S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H₂S. In conclusion, lung protection by H₂S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H₂S mediated protection.     

Histometric features for the grading of prostatic carcinoma

Histometric features for the objective grading of prostatic adenocarcinoma in histologic specimens were analyzed in five cases each of well, moderately and poorly differentiated lesions. Tissue sections from the selected cases were stained by the Feulgen method and digitized by a video-based microphotometer. Twenty total fields were recorded for each grade: ten at high resolution (an image sampling of 0.5 micron per pixel) and ten at low resolution (0.8 micron per pixel), with two fields per case recorded at each resolution. The images were segmented by an automated expert system-guided scene segmentation procedure. The performance of that procedure was measured by comparing the automated counts of nuclei in the segmented fields to the visual counts made by a pathologist in the same fields. For well, moderately and poorly differentiated cases, respectively, the nuclear counts made by the expert system at high resolution were 2.7%, 4.2% and 4.7% higher than the visual counts (as estimated from a total of 6,628 nuclei), but 1.2%, 2.5% and 1.1% lower at low resolution (10,329 nuclei). High-resolution features and tissue textural features were computed for each case. The high-resolution features showed good separation between the three groups of cases. The tissue textural features showed consistent separation between well and moderately differentiated cases. The relaxation of the spatial resolution (to 0.8 micron/pixel spacing) did not affect the selection of features, but led to less separation between the data from different grades. In conclusion, the automated system performed satisfactorily in distinguishing sections of prostatic tumors of varying degrees of differentiation.     

Climate change alters plant biogeography in Mediterranean prairies along the West Coast,USA

Projected changes in climate are expected to have widespread effects on plant community composition and diversity in coming decades. However, multisite, multifactor climate manipulation studies that have examined whether observed responses are regionally consistent and whether multiple climate perturbations are interdependent are rare. Using such an experiment, we quantified how warming and increased precipitation intensity affect the relative dominance of plant functional groups and diversity across a broad climate gradient of Mediterranean prairies. We implemented a fully factorial climate manipulation of warming (+2.5–3.0 °C) and increased wet‐season precipitation (+20%) at three sites across a 520‐km latitudinal gradient in the Pacific Northwest, USA. After seeding with a nearly identical mix of native species at all sites, we measured plant community composition (i.e., cover, richness, and diversity), temperature, and soil moisture for 3 years. Warming and the resultant drying of soils altered plant community composition, decreased native diversity, and increased total cover, with warmed northern communities becoming more similar to communities further south. In particular, after two full years of warming, annual cover increased and forb cover decreased at all sites mirroring the natural biogeographic pattern. This suggests that the extant climate gradient of increasing heat and drought severity is responsible for a large part of the observed biogeographic pattern of increasing annual invasion in US West Coast prairies as one moves further south. Additional precipitation during the rainy season did little to relieve drought stress and had minimal effects on plant community composition. Our results suggest that the projected increase in drought severity (i.e., hotter, drier summers) in Pacific Northwest prairies may lead to increased invasion by annuals and a loss of forbs, similar to what has been observed in central and southern California, resulting in novel species assemblages and shifts in functional composition, which in turn may alter ecosystem functions.     

Gradient-based optimization of kernel-target alignment for sequence kernels applied to bacterial gene start detection

Biological data mining using kernel methods can be improved by a task-specific choice of the kernel function. Oligo kernels for genomic sequence analysis have proven to have a high discriminative power and to provide interpretable results. Oligo kernels that consider subsequences of different lengths can be combined and parameterized to increase their flexibility. For adapting these parameters efficiently, gradient-based optimization of the kernel-target alignment is proposed. The power of this new, general model selection procedure and the benefits of fitting kernels to problem classes are demonstrated by adapting oligo kernels for bacterial gene start detection     

The ultimate and proximate mechanisms driving the evolution of long tails in forest deer mice

Understanding both the role of selection in driving phenotypic change and its underlying genetic basis remain major challenges in evolutionary biology. Here, we use modern tools to revisit a classic system of local adaptation in the North American deer mouse, Peromyscus maniculatus, which occupies two main habitat types: prairie and forest. Using historical collections, we find that forest‐dwelling mice have longer tails than those from nonforested habitat, even when we account for individual and population relatedness. Using genome‐wide SNP data, we show that mice from forested habitats in the eastern and western parts of their range form separate clades, suggesting that increased tail length evolved independently. We find that forest mice in the east and west have both more and longer caudal vertebrae, but not trunk vertebrae, than nearby prairie forms. By intercrossing prairie and forest mice, we show that the number and length of caudal vertebrae are not correlated in this recombinant population, indicating that variation in these traits is controlled by separate genetic loci. Together, these results demonstrate convergent evolution of the long‐tailed forest phenotype through two distinct genetic mechanisms, affecting number and length of vertebrae, and suggest that these morphological changes—either independently or together—are adaptive.