The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. To investigate the relative contribution of genomic and epigenetic modifications towards phenotype evolution, comprehensive genome and epigenome data are presented for six related CHO cell lines, both in response to perturbations (different culture conditions and media as well as selection of a specific phenotype with increased transient productivity) and in steady state (prolonged time in culture under constant conditions). Clear transitions were observed in DNA‐methylation patterns upon each perturbation, while few changes occurred over time under constant conditions. Only minor DNA‐methylation changes were observed between exponential and stationary growth phase; however, throughout a batch culture the histone modification pattern underwent continuous adaptation. Variation in genome sequence between the six cell lines on the level of SNPs, InDels, and structural variants is high, both upon perturbation and under constant conditions over time. The here presented comprehensive resource may open the door to improved control and manipulation of gene expression during industrial bioprocesses based on epigenetic mechanisms. Biotechnol. Bioeng. 2016;113: 2241–2253. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Escherichia coli K–12 and B strains are among the most frequently used bacterial hosts for production of recombinant proteins on an industrial scale. To improve existing processes and to accelerate bioprocess development, we performed a detailed host analysis. We investigated the different behaviors of the E. coli production strains BL21, RV308, and HMS174 in response to high-glucose concentrations. Tightly controlled cultivations were conducted under defined environmental conditions for the in-depth analysis of physiological behavior. In addition to acquisition of standard process parameters, we also used DNA microarray analysis and differential gel electrophoresis (EttanTM DIGE). Batch cultivations showed different yields of the distinct strains for cell dry mass and growth rate, which were highest for BL21. In addition, production of acetate, triggered by excess glucose supply, was much higher for the K–12 strains compared to the B strain. Analysis of transcriptome data showed significant alteration in 347 of 3882 genes common among all three hosts. These differentially expressed genes included, for example, those involved in transport, iron acquisition, and motility. The investigation of proteome patterns additionally revealed a high number of differentially expressed proteins among the investigated hosts. The subsequently selected 38 spots included proteins involved in transport and motility. The results of this comprehensive analysis delivered a full genomic picture of the three investigated strains. Differentially expressed groups for targeted host modification were identified like glucose transport or iron acquisition, enabling potential optimization of strains to improve yield and process quality. Dissimilar growth profiles of the strains confirm different genotypes. Furthermore, distinct transcriptome patterns support differential regulation at the genome level. The identified proteins showed high agreement with the transcriptome data and suggest similar regulation within a host at both levels for the identified groups. Such host attributes need to be considered in future process design and operation.

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision.-Morak, M., H. Schmidinger, P. Krempl, G. Rechberger, M. Kollroser, R. Birner-Gruenberger, and A. Hermetter. Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities. In the postgenomic era, researchers are now challenging the proteome with methods like two-dimensional gel electrophoresis or multidimensional chromatography followed by mass spectrometry and various other methods for abundance-based proteome profiling. However, the amount of proteins present at a certain state of the cell might not correlate with the enzyme activities responsible for the metabolic fluxes, cell management, and signal transduction. Therefore, elucidation of changes in protein activity is the ultimate goal of functional proteomics.Methods have been developed for specific detection of enzymes on the basis of their catalytic activities. For this purpose, activity recognition probes (ARPs) (1) or activitybased probes are used. Basically, an ARP is a molecule consisting of i) a recognition site targeting a certain enzyme species, ii) a properly positioned reactive site that forms a covalent bond with the target, and iii) a tag for visualization and/or isolation of the covalently bound target (2-4). Many reactive groups have been developed so far for identifying different types of enzyme activity, i.e., fluorophosphonates for serine hydrolases (5), p-nitrophenyl organophosphonates for lipases and esterases (3, 6), epoxides for cysteine proteases (7), and sulfonate esters for various enzyme classes (8, 9). Two-dimensional (2D) gel electrophoresis is a well-established technique for simultaneous separation and display of hundreds to thousan...

The endophytic bacterium Pseudomonas poae RE*1-1-14 shows broad antagonistic activity and is applied to seeds as a biocontrol agent to suppress late root rot in the sugar beet. The completely sequenced 5.5-Mb genome reveals genes that putatively contribute to this antagonistic activity and the intimate plant-microbe interaction.