Biology subjects

Silver, P. A.

Publications and source records attributed to Silver, P. A..

43 records · Page 3Linked to original sources

bioRxiv2018

Mammalian Cells Engineered to Produce Novel Steroids

Steroids can be difficult to modify via traditional organic synthesis methods, but many enzymes regio- and stereo-selectively process a wide variety of steroid substrates. We tested whether steroid-modifying enzymes could make novel steroids from non-native substrates. Numerous genes encoding steroid-modifying enzymes, including some bacterial enzymes, were expressed in mammalian cells by transient transfection and found to be active. We made three unusual steroids by expression in HEK293 cells of the 7-hydroxylase CYP7B1, which was selected because of high native product yield. These cells made 7,17-dihydroxypregnenolone and 7{beta},17-dihydroxypregnenolone from 17-hydroxypregnenolone, and produced 11,16-dihydroxyprogesterone from 16-hydroxyprogesterone. The latter two products resulted from previously unobserved CYP7B1 hydroxylation sites. A Rosetta docking model of CYP7B1 suggested that these substrates D-ring hydroxylations may prevent them from binding in the same way as the native substrate, bringing different carbons near the active ferryl oxygen. This new approach could use other enzymes and substrates to produce many novel steroids for drug candidate testing.

synthetic biology↗

bioRxiv2018

Prokaryotic nanocompartments form synthetic organelles in a eukaryote

Compartmentalization of proteins into organelles is a promising strategy for enhancing the productivity of engineered eukaryotic organisms. However, approaches that co-opt endogenous organelles may be limited by the potential for unwanted crosstalk and disruption of native metabolic functions. Here, we present the construction of synthetic non-endogenous organelles in the eukaryotic yeast Saccharomyces cerevisiae, based on the prokaryotic family of self-assembling proteins known as encapsulins. We establish that encapsulins self-assemble to form nanoscale compartments in yeast, and that heterologous proteins can be selectively targeted for compartmentalization. Housing destabilized proteins within encapsulin compartments affords protection against proteolytic degradation in vivo, while the interaction between split protein components is enhanced upon co-localization within the compartment interior. Furthermore, encapsulin compartments can support enzymatic catalysis, with substrate turnover observed for an encapsulated yeast enzyme. Encapsulin compartments therefore represent a modular platform, orthogonal to existing organelles, for programming synthetic compartmentalization in eukaryotes.

synthetic biology↗

bioRxiv2017

Rational Design of Evolutionarily Stable Microbial Kill Switches

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One particularly useful but challenging situation is a switch between life and death depending on environment. Here are presented \"essentializer\" and \"cryodeath\" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a novel toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. In addition, cryodeath was shown to control the growth environment of a bacterial population, with an escape rate of less than 1 in 105 after ten days in vivo.

synthetic biology↗

bioRxiv2017

Rapid genome recoding by iterative recombineering of synthetic DNA

Genome recoding will provide a deeper understanding of genetics and transform biotechnology. We bypass the reliance of previous genome recoding methods on site-specific enzymes and demonstrate a rapid recombineering based strategy for writing genomes by Stepwise Integration of Rolling Circle Amplified Segments (SIRCAS). We installed the largest number of codon substitutions in a single organism yet published, creating a strain of Salmonella typhimurium with 1557 leucine codon changes across 200 kb of the genome.

synthetic biology↗

bioRxiv2016

Super-resolution microscopy of the β-carboxysome reveals a homogenous matrix

Carbon fixation in cyanobacteria makes a major contribution to the global carbon cycle. The cyanobacterial carboxysome is a proteinaceous microcompartment that protects and concentrates the carbon-fixing enzyme RuBisCO in a paracrystalline lattice, making it possible for these organisms to fix CO2 from the atmosphere. The protein responsible for the organization of this lattice in beta-type carboxysomes of the freshwater cyanobacterium Synechococcus elongatus, CcmM, occurs in two isoforms thought to localize differentially within the carboxysome matrix. Here we use widefield timelapse and 3D-structured illumination microscopy (3D-SIM) to study the recruitment and localization of these two isoforms. We demonstrate that this super-resolution technique is capable of successfully resolving the outer protein shell of the carboxysome from its internal cargo. We develop an automated analysis pipeline to analyze and quantify 3D-SIM images and generate a population level description of carboxysome shell protein, RuBisCO, and CcmM isoform localization. We find that both CcmM isoforms colocalize in space and time, prompting a revised model of the internal arrangement of the beta carboxysome.

cell biology↗

bioRxiv2016

Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution

Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.

synthetic biology↗

bioRxiv2016

Microbes use encapsulin protein organelles to sequester toxic reactions

Cells organize and regulate their metabolism via membrane-or protein-bound organelles. In this way, incompatible processes can be spatially separated and controlled. In prokaryotes, protein-based compartments are used to sequester harmful reactions and store useful compounds. These protein compartments play key roles in various metabolic and ecological processes ranging from iron homeostasis to carbon fixation. We identified more than 900 proteinaceous encapsulin nanocompartment systems in bacterial and archaeal genomes. Encapsulins can be found in 15 bacterial and 2 archaeal phyla. Our analysis reveals 1 new capsid type and 9 previously unknown cargo proteins targeted to the interior of encapsulins. We experimentally characterize 3 newly identified encapsulin systems and illustrate their involvement in iron mineralization, oxidative and nitrosative stress resistance and anaerobic ammonium oxidation, a process responsible for 30% of the N lost from the oceans. We propose that encapsulins represent a widespread strategy for toxic reaction sequestration in prokaryotes.

biochemistry↗