Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase.

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α6-ß6S. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.

Engineering Cellular Metabolism.

Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation.

Deciphering the principles of the RNA editing code via large-scale systematic probing.

Adenosine-to-inosine editing is catalyzed by ADAR1 at thousands of sites transcriptome-wide. Despite intense interest in ADAR1 from physiological, bioengineering, and therapeutic perspectives, the rules of ADAR1 substrate selection are poorly understood. Here, we used large-scale systematic probing of ∼2,000 synthetic constructs to explore the structure and sequence context determining editability. We uncover two structural layers determining the formation and propagation of A-to-I editing, independent of sequence. First, editing is robustly induced at fixed intervals of 35 bp upstream and 30 bp downstream of structural disruptions. Second, editing is symmetrically introduced on opposite sites on a double-stranded structure. Our findings suggest a recursive model for RNA editing, whereby the structural alteration induced by the editing at one site iteratively gives rise to the formation of an additional editing site at a fixed periodicity, serving as a basis for the propagation of editing along and across both strands of double-stranded RNA structures.

Membrane protein synthesis: no cells required.

Despite advances in membrane protein (MP) structural biology and a growing interest in their applications, these proteins remain challenging to study. Progress has been hindered by the complex nature of MPs and innovative methods will be required to circumvent technical hurdles. Cell-free protein synthesis (CFPS) is a burgeoning technique for synthesizing MPs directly into a membrane environment using reconstituted components of the cellular transcription and translation machinery in vitro. We provide an overview of CFPS and how this technique can be applied to the synthesis and study of MPs. We highlight numerous strategies including synthesis methods and folding environments, each with advantages and limitations, to provide a survey of how CFPS techniques can advance the study of MPs.

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Genetic knockdown of genes that are obscure, conserved and essential using CRISPR interference methods in the fission yeast S. pombe.

Characterizing functions of essential genes is challenging, as perturbing them is generally lethal. Conditional gene perturbation, including use of temperature-sensitive mutants, has been widely utilized to reveal functions of essential genes in the fission yeast Schizosaccharomyces pombe. However, recently we implemented a systematic and less time-consuming knockdown method, CRISPR interference (CRISPRi), in this organism using catalytically inactive Cas9 (dCas9). This technology has been expected to facilitate characterization of essential genes in S. pombe, although this still has not occurred. Here, CRISPRi was harnessed to study uncharacterized essential genes that are evolutionally conserved from yeasts to mammals. Transcription of these genes, which we call conserved essential obscure (ceo) genes, was repressed using conventional dCas9-mediated CRISPRi and by implementing technologies that enhance repression efficiency or alleviate limitations on small guide RNA (sgRNA) design. These CRISPRi methods successfully reduced transcription of target genes and allowed us to characterize resulting phenotypes. Knockdown of ceo genes inhibited cell proliferation and altered cellular morphology. Thus, dCas9-based CRISPRi methods utilized in this study enhanced accessibility of genetic analyses targeting essential genes in S. pombe.

Integration of Agrobacterium T-DNA into the Plant Genome.

Agrobacterium strains transfer a single-strand form of T-DNA (T-strands) and Virulence (Vir) effector proteins to plant cells. Following transfer, T-strands likely form complexes with Vir and plant proteins that traffic through the cytoplasm and enter the nucleus. T-strands may subsequently randomly integrate into plant chromosomes and permanently express encoded transgenes, a process known as stable transformation. The molecular processes by which T-strands integrate into the host genome remain unknown. Although integration resembles DNA repair processes, the requirement of known DNA repair pathways for integration is controversial. The configuration and genomic position of integrated T-DNA molecules likely affect transgene expression, and control of integration is consequently important for basic research and agricultural biotechnology applications. This article reviews our current knowledge of the process of T-DNA integration and proposes ways in which this knowledge may be manipulated for genome editing and synthetic biology purposes.

Microbes as Biosensors.

The ability to detect disease early and deliver precision therapy would be transformative for the treatment of human illnesses. To achieve these goals, biosensors that can pinpoint when and where diseases emerge are needed. Rapid advances in synthetic biology are enabling us to exploit the information-processing abilities of living cells to diagnose disease and then treat it in a controlled fashion. For example, living sensors could be designed to precisely sense disease biomarkers, such as by-products of inflammation, and to respond by delivering targeted therapeutics in situ. Here, we provide an overview of ongoing efforts in microbial biosensor design, highlight translational opportunities, and discuss challenges for enabling sense-and-respond precision medicines.

A comprehensive preanalytical protocol for fresh solid tumor biospecimens.

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.