Primary and Secondary Metabolic Effects of a Key Gene Deletion (ΔYPL062W) in Metabolically Engineered Terpenoid-ProducingSaccharomyces cerevisiae

ABSTRACTSaccharomyces cerevisiaeis an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion ofYPL062WinS. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function ofYPL062Wand the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered thatYPL062Wdeletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed thatYPL062Wfunctions as a core promoter forALD6and that the expression level ofALD6is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062Wincreases the production of all major terpenoid classes (C10, C15, C20, C30, and C40). Our study not only elucidated the biological function ofYPL062Wbut also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCEAlthough computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms ofYPL062Wdeletion inS. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function ofYPL062W, demonstrates a strong correlation betweenYPL062Wand terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.

QSAR and Lead Optimization

There are many diseases for which suitable drugs have not been identified. As the population increases and the environment gets polluted, new infections are reported. Random screening of synthesized compounds for biological activity is time consuming. QSAR has a prominent role in drug design and optimization. It is derived from the correlation between the physicochemical properties and biological activity. QSAR equations are generated using statistical methods like regression analysis and genetic function approximation. Both 2D parameters and 3D parameters are involved in generating the equation. Among several QSAR equations generated, the best ones are selected based on statistical parameters. Validation techniques usually verify the predictive power of generated QSAR equations. Once the developed QSAR model is validated to be good, the results of that model may be applied to predict the biological activity of newer analogues. This chapter illustrates the various steps in QSAR and describes the significance of statistical parameters and software used in QSAR.

Epigenetics

The field of Epigenetics represents the science which helps us understand the influence life has on genetic function. Research has shown that what we do, what we are exposed to, and the internal and external environments have significant influence on the output of our genes. In this article, the reader will understand how alterations in the DNA explain how the genetic output us changed as a result of exposures to life.

Revisiting Plozévet

A special issue of French Cultural Studies in 1999 sought to explore the ways in which Modern Linguists have often tended to erase traces of their personal lives from their work. Contributors responded in an autobiographical mode by exploring the ‘hidden selves of scholars and teachers’. This article builds on these reflections by exploring the extent to which Edgar Morin’s Commune en France (1967), his contribution to the multidisciplinary project in Plozévet in Brittany, may be understood as ‘autobiographie involontaire’. The study reads Morin’s Journal de Plozévet (published in 2001, over three decades after the research was completed) in relation to the original monograph and suggests that the diary operates as the second panel of a diptych that reveals the texts’ interdependence. The journal fulfils a ‘genetic’ function, providing the sources for elements of Morin’s monograph and giving an indication of the extent to which Morin drew on the work of his wider team to complement his own observations. More importantly, however, reading the two texts in counterpoint – the one providing spontaneous reflections in the field; the other revealing their processing in the immediate aftermath of the enquête – is part of the revelation of the ‘hidden selves’ of a researcher embedded in a monograph that has been long considered a classic of post-war French sociology.

Adaptive strategies of the candidate probioticE. coliNissle in the mammalian gut

SummaryProbiotics are living microorganisms that are increasingly used as gastrointestinal therapeutics by virtue of their innate or engineered genetic function. Unlike abiotic therapeutics, probiotics can replicate in their intended site, subjecting their genomes and therapeutic properties to natural selection. By exposing the candidate probioticE. coliNissle (EcN) to the mouse gastrointestinal tract over several weeks, we uncovered the consequences of gut transit, inter-species competition, antibiotic pressure, and engineered genetic function on the processes under selective pressure during both within-genome and horizontal evolutionary modes. We then show the utility of EcN as a chassis for engineered function by achieving the highest reported reduction in serum phenylalanine levels in a mouse model of phenylketonuria using an engineered probiotic. Collectively, we demonstrate a generalizable pipeline which can be applied to other probiotic strains to better understand their safety and engineering potential.