Optimized Hemolysin Type 1 Secretion System in Escherichia coli by Directed Evolution of the Hly Enhancer Fragment and Including a Terminator Region

Type 1 secretion systems (T1SS) have a relatively simple architecture compared to other classes of secretion systems and therefore, are attractive to be optimized by protein engineering. Here, we report a KnowVolution campaign for the hemolysin (Hly) enhancer fragment, an untranslated region upstream of the hlyA gene, of the hemolysin T1SS of Escherichia coli to enhance its secretion efficiency. The best performing variant of the Hly enhancer fragment contained five nucleotide mutations at five positions (A30U, A36U, A54G, A81U, and A116U) resulted in a 2-fold increase in the secretion level of a model lipase fused to the secretion carrier HlyA1. Computational analysis suggested that altered affinity to the generated enhancer fragment towards the S1 ribosomal protein contributes to the enhanced secretion levels. Furthermore, we demonstrate that involving a native terminator region along with the generated Hly enhancer fragment increased the secretion levels of the Hly system up to 5-fold.     

A 1D numerical model for rapid stress analysis in bipolar junction transistors

Stress effects in semiconductor devices have gained significant attention in semiconductor industry in recent years, and numerical modeling is often used as a powerful tool for stress analysis in semiconductor devices. Here, we present a nontraditional 1D model for fast stress analysis in bipolar junction transistors. Because bipolar transistors are operationally 1D devices, it is possible to speed up the simulation with a 1D numerical model and get results that are comparable with 2D and 3D simulation outcomes. This model consists of a complete numerical algorithm that can be used for stress analysis of bipolar transistors on any plane. Existing 1D simulators take more time as they solve all device equations throughout the device. In contrast, our model optimizes the solutions for different regions with the development and inclusion of specific algorithms. A fractional starting point is introduced for the depletion region to speed up the process further. This way, faster computing time and much higher accuracy can be reached. At the same time, popular 2D and 3D simulators, which are using finite element methods, are naturally much slower, especially if high accuracy is needed. Simulation results of this 1D model match well with the simulation results of a 2D model developed with a commercial technology computer aided design (TCAD) tool. The validity of our model was verified with experimental results and theoretical expectations as well. Copyright © 2016 John Wiley & Sons, Ltd.     

Biotransformation Of l-Tryptophan To Produce Arcyriaflavin A With Pseudomonas putida KT2440

Natural products such as indolocarbazoles are a valuable source of highly bioactive compounds with numerous potential applications in the pharmaceutical industry. Arcyriaflavin A, isolated from marine invertebrates and slime molds, is one representative of this group and acts as a cyclin D1-cyclin-dependent kinase 4 inhibitor. To date, access to this compound has mostly relied on multi-step total synthesis. In this study, biosynthetic access to arcyriaflavin A was explored using recombinant Pseudomonas putida KT2440 based on a previously generated producer strain. We used a Design of Experiment approach to analyze four key parameters, which led to the optimization of the bioprocess. By engineering the formation of outer membrane vesicles and using an adsorbent in the culture broth, we succeeded to increase the yield of arcyriaflavin A in the cell-free supernatant, resulting in a nearly eight-fold increase in the overall production titers. Finally, we managed to scale up the bioprocess leading to a final yield of 4.7 mg arcyriaflavin A product isolated from 1 L of bacterial culture. Thus, this study showcases an integrative approach to improve biotransformation and moreover also provides starting points for further optimization of indolocarbazole production in P. putida.     

Carrier-Free Enzyme Immobilizates for Flow Chemistry

For the development of efficient and green industrial processes, the combination of biocatalysis and flow chemistry holds great promises. Flow chemical utilization of biocatalysts, essentially made possible by the immobilization (or retention) of enzymes in flow reactors, has attracted increased academic attention during recent years. In the present review we present an overview of immobilization strategies suitable for flow chemistry, particularly focusing on recently developed carrier-free immobilization methods, highlighting advances in the field and presenting future trends.