A semisynthetic organism engineered for the stable expansion of the genetic alphabet

Zhang, Yorke; Lamb, Brian M.; Feldman, Aaron W.; Zhou, Anne X-Z; Lavergne, Thomas; Li, Lingjun; Romesberg, Floyd E.

doi:10.1073/pnas.1616443114

Cited by 153 publications

(213 citation statements)

References 42 publications

(33 reference statements)

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“…3, 127, 129, 137–139 To date, ncAAs have been employed in a large number of studies of protein structure and function, and also to alter or enhance protein function. In addition, the recombinant introduction of ncAAs into proteins is allowing the development of new therapeutics and diagnostics.…”

Section: Applications Of Non-canonical Amino Acidsmentioning

confidence: 99%

Playing with the Molecules of Life

2018

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Our understanding of the complex processes of living organisms at the molecular level is growing exponentially. This knowledge, together with a powerful arsenal of tools for manipulating the structures of macromolecules, is allowing chemists to harness and reprogram the cellular machinery. Here we review one example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins in ways previously unimaginable

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Section: Applications Of Non-canonical Amino Acidsmentioning

confidence: 99%

Playing with the Molecules of Life

2018

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“…A very striking one includes the cloning and engineering of an NTP transporter to allow E coli to take up non-natural nucleotide triphosphates, to encode "a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet" [329]. As to efflux transporters, the engineering towards influx of substances normally pumped out, for instance via uncoupled variants of the LmrP 'efflux' transporter in lactobacilli [330][331][332][333] is notable.…”

Section: Directed Evolution: Changing the Sequence Of The Target Tranmentioning

confidence: 99%

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Kell¹

2018

Preprint

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The yield and ease of purification of biotechnological products are typically greatly enhanced if they can be secreted into the external medium. Although not universally appreciated, however, small molecule biotechnological products do not ‘float across’ the phospholipid bilayer portion of biological membranes, and thus they need transporters to assist their passage into the extramembrane and extracellular spaces. Some of these transporters may be reversible, equilibrative transporters that might more normally be used for uptake, while others may have an efflux directionality imposed on them via suitable energy coupling mechanisms. Despite the energetic costs of small molecule synthesis, natural evolution does in fact provide a number of mechanisms by which the secretion of such products can actually enhance fitness. Where available these provide useful starting points, and assaying for such activities is crucial. In particular, in a systems biology approach, we first need to identify such/suitable activities before we can seek to increase them. They may then be improved through promoter engineering, via manipulation of control elements, or by directed evolution of the transporter proteins themselves. Modern methods of synthetic biology provide enormous opportunities for all kinds of efflux transporter engineering; they are just beginning to be realised.

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“…5 However, unlike in vitro replication, replication in this SSO showed significant sequence biases, some of which were still observed with the d NaM -d TPT3 UBP. 6 This is perhaps not surprising considering that the in vivo replication environment is distinct from the in vitro environment, which had been used to generate the SAR that drove UBP optimization and standardization. Thus, we screened 135 candidate UBPs, drawn from 91 unnatural triphosphates selected to cover the range of analogs that had been explored in vitro , for those that when added to the media were able to support high level retention of the corresponding UBP on a plasmid within the SSO.…”

Section: In Vivo Performance and Optimizationmentioning

confidence: 99%

Expansion of the Genetic Alphabet: A Chemist’s Approach to Synthetic Biology

Feldman

Romesberg

2017

Acc. Chem. Res.

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ConspectusThe information available to any organism is encoded in a four nucleotide, two base pair genetic code. Since its earliest days, the field of synthetic biology has endeavored to impart organisms with novel attributes and functions, and perhaps the most fundamental approach to this goal is the creation of a fifth and sixth nucleotide that pair to form a third, unnatural base pair (UBP) and thus allow for the storage and retrieval of increased information. Achieving this goal, by definition, requires synthetic chemistry to create unnatural nucleotides and a medicinal chemistry-like approach to guide their optimization. With this perspective, almost 20 years ago we began designing unnatural nucleotides with the ultimate goal of developing UBPs that function in vivo, and thus serve as the foundation of semi-synthetic organisms (SSOs) capable of storing and retrieving increased information. From the beginning, our efforts focused on the development of nucleotides that bear predominantly hydrophobic nucleobases and thus that pair not based on the complementary hydrogen bonds that are so prominent among the natural base pairs but rather via hydrophobic and packing interactions. It was envisioned that such a pairing mechanism would provide a basal level of selectivity against pairing with natural nucleotides, which we expected would be the greatest challenge; however, this choice mandated starting with analogs that have little or no homology to their natural counterparts and that, perhaps not surprisingly, performed poorly. Progress toward their optimization was driven by the construction of structure–activity relationships, initially from in vitro steady-state kinetic analysis, then later from pre-steady-state and PCR-based assays, and ultimately from performance in vivo, with the results augmented three times with screens that explored combinations of the unnatural nucleotides that were too numerous to fully characterize individually. The structure–activity relationship data identified multiple features required by the UBP, and perhaps most prominent among them was a substituent ortho to the glycosidic linkage that is capable of both hydrophobic packing and hydrogen bonding, and nucleobases that stably stack with flanking natural nucleobases in lieu of the potentially more stabilizing stacking interactions afforded by cross strand intercalation. Most importantly, after the examination of hundreds of unnatural nucleotides and thousands of candidate UBPs, the efforts ultimately resulted in the identification of a family of UBPs that are well recognized by DNA polymerases when incorporated into DNA and that have been used to create SSOs that store and retrieve increased information. In addition to achieving a longstanding goal of synthetic biology, the results have important implications for our understanding of both the molecules and forces that can underlie biological processes, so long considered the purview of molecules benefiting from eons of evolution, and highlight the promise of applying the approaches an...

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A semisynthetic organism engineered for the stable expansion of the genetic alphabet

Cited by 153 publications

References 42 publications

Playing with the Molecules of Life

Playing with the Molecules of Life

Control of metabolite efflux in microbial cell factories: current advances and future prospects

Expansion of the Genetic Alphabet: A Chemist’s Approach to Synthetic Biology

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