Enhanced Butanol Production in Clostridium acetobutylicum Using Small Regulatory RNAs for Metabolic Engineering

This project uses an RNA-based gene expression control system for metabolic engineering of Clostridium acetobutylicum. The primary focus is enhancing production of n-butanol, a green alternative to fossil fuels. Butanol is an attractive alternative fuel with higher energy density than other biofuels, and can directly replace gasoline. To improve the production of butanol using Clostridium, a small RNA (sRNA) platform is utilized. RNA is useful as a genetic regulatory tool because it provides flexible expression tuning compared to the on/off DNA knockout method. sRNAs are used by bacteria for regulating gene expression, as they bind to protein-coding mRNA sequences. Through binding, sRNA can enhance or reduce mRNA translation and thus protein expression. Two genes in the metabolic pathway, buk and hydA, will be down regulated using sRNA, potentially increasing butanol titer and yield without compromising cell growth. Down-regulation of these genes presents a novel opportunity to modify Clostridium, as hydA is essential to cellular function and cannot be completely turned off. Furthermore, using sRNA allows for simultaneous targeting of both genes. This is done using genetic engineering techniques to transform wild type cells with the desired genes. The recombinant plasmid for sRNA production is derived from E. coli and then ported over. Mutants are then screened and tested to determine performance as compared to the original strain. One mutant and a control plasmid have been successfully transformed. Preliminary results show that the mutant targeting hydA successfully down regulates the gene and reduces production of butyric acid, while also increasing butanol production. This project could have significant contribution to improving economic viability of biologically derived n-butanol. Furthermore, the sRNA platform has potential for broad applications in metabolically engineering various bacterial species.NSFNo embargoAcademic Major: Chemical Engineerin

Optimizing a bacterial sRNA scaffold for targeting multiple mRNAs, filtering off- target mRNA interactions, and balancing metabolic pathway flux

RNA is central to gene expression control in cells, and yet the small regulatory RNAs (sRNAs) of bacteria are still in the early stages of development as synthetic biology tools. The ubiquity and diversity of sRNAs in bacteria bodes well for engineering synthetic sRNA control of metabolic pathways, particularly in organisms with poorly developed genetic tools. These sRNAs regulate mRNA targets by RNA:RNA base-pairing interactions, and sRNAs have been retargeted to regulate non-native mRNAs for metabolic engineering applications.1-3 However, an ongoing concern about sRNAs as tools is their potential for hybridizing to off-target mRNAs. Here we describe the development, optimization and implementation of a structured sRNA scaffold with improved target discrimination relative to an unstructured antisense sRNA scaffold. Native DsrA sRNA4,5 contains two striking stem-loop antisense motifs that use antisense base-pairing to coordinately regulate translation of two E. coli mRNA targets. Previously6 we created a genetic system for retargeting DsrA simultaneously to two non-native mRNA targets in E. coli. Next, we expressed a retargeted E. coli sRNA variant in C. acetobutylicum cultures to improve n-butanol biofuel fermentation yield and selectivity. We achieved this goal by retargeting a DsrA sRNA variant to tune-down expression of an essential clostridial hydrogenase and increase NADH levels in the fermentation culture. Finally, we used this E. coli sRNA genetic system to demonstrate that the stem-loop antisense “fingerloop” structures can be configured to exclude certain off-target mRNA interactions. This fingerloop antisense motif constitutes a very promising programmable target–mRNA control element that is modular, discriminating, and portable between organisms. Since fine-tuning and balancing metabolic pathway flux is an important scale-up parameter, this nanoscale sRNA tool should be particularly useful in industrial scale bacterial fermentations of biofuels and specialty chemicals. Please click Additional Files below to see the full abstract

Application of urine proteomics for biomarker discovery in drug-induced liver injury

Abstract The leading cause of hepatic damage is drug-induced liver injury (DILI), for which currently no adequate predictive biomarkers are available. Moreover, for most drugs related to DILI, the mechanisms underlying the adverse reaction have not yet been elucidated. Urinary protein biomarker candidates for DILI have emerged in the past few years and correlate well with clinical studies for serum DILI biomarkers. The goal of this review was to investigate the use of urine as a source of protein biomarkers for drug-induced liver injury. Finally, we discuss some of the current strategies required to advance the field of biomarker discovery for DILI with respect to appropriate clinical biobanking and adequate translational research