Synthetic Biology

Resurrecting essential amino acid biosynthesis in mammalian cells

1. Julie Trolle
2. Ross M McBee
3. Andrew Kaufman
4. Sudarshan Pinglay
5. Henri Berger
6. Sergei German
7. Liyuan Liu
8. Michael J Shen
9. Xinyi Guo
10. J Andrew Martin
11. Michael E Pacold
12. Drew R Jones
13. Jef D Boeke
14. Harris H Wang

• Curated by eLife

  Evaluation Summary:

  In this study, Trolle et al aimed to introduce methionine, threonine, isoleucine, and valine biosynthetic pathways into Chinese Hamster Ovary (CHO) cells. While this was unsuccessful for methionine, threonine, and isoleucine, introduction of valine synthesis rendered CHO cells partially independent on exogenous valine. Although introduction of essential amino acid biosynthetic pathways into mammalian cells is of potentially broad interest to the fields of synthetic biology, biotechnology and metabolism, there were concerns regarding incomplete demonstration that the introduction of valine pathway into CHO cells is sufficient to sustain homeostasis in the absence of exogenous valine. Further metabolic/biochemical characterization of valine-producing CHO cells is warranted.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

Reviewed by eLife

A Pan-Coronavirus Vaccine Candidate: Nine Amino Acid Substitutions in the ORF1ab Gene Attenuate 99% of 365 Unique Coronaviruses: A Comparative Effectiveness Research Study

1. Eric Luellen

Reviewed by ScreenIT

De novo-designed transmembrane domains tune engineered receptor functions

1. Assaf Elazar
2. Nicholas J Chandler
3. Ashleigh S Davey
4. Jonathan Y Weinstein
5. Julie V Nguyen
6. Raphael Trenker
7. Ryan S Cross
8. Misty R Jenkins
9. Melissa J Call
10. Matthew E Call
11. Sarel J Fleishman

• Curated by eLife

  Evaluation Summary:

  This is an interesting paper that uses de novo protein design to probe the effects of oligomerization state on the activity of chimeric antigen receptors (CARS). The successful design of transmembrane domains with specific oligomeric states is an impressive result on its own. The proteins were designed using rotamer-based sequence optimization in Rosetta with an energy function specific for the membrane environment. After experimentally evaluating a couple rounds of designs, the investigators settled on a design protocol that also included screening of the design candidates with docking simulations in alternative oligomerization states to check that the sequences preferred the desired oligomerization state. The designs were experimentally evaluated with gel electrophoresis and X-ray crystallography. In the end, designs that adopted well-defined dimers, trimers, or tetramers were created and carried forward in experiments as CARs.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

Reviewed by eLife

Accelerating PERx reaction enables covalent nanobodies for potent neutralization of SARS-CoV-2 and variants

1. Bingchen Yu
2. Shanshan Li
3. Takako Tabata
4. Nanxi Wang
5. Li Cao
6. G. Renuka Kumar
7. Wei Sun
8. Jun Liu
9. Melanie Ott
10. Lei Wang

Reviewed by ScreenIT

Dynamic spreading of chromatin-mediated gene silencing and reactivation between neighboring genes in single cells

1. Sarah Lensch
2. Michael H Herschl
3. Connor H Ludwig
4. Joydeb Sinha
5. Michaela M Hinks
6. Adi Mukund
7. Taihei Fujimori
8. Lacramioara Bintu

• Curated by eLife

  Evaluation Summary:

  This study describes a novel approach to investigate how the transcriptional repressors KRAB and HDAC4 repress gene expression, how repression spreads over differing genomic distances, and what the role of insulator elements is in blocking the spread of repression and in reactivation of repressed genes. The results of this study allow modeling of the coordinated repression or activation of closely linked genes and should be of wide interest to researchers interested in chromatin and gene expression.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

Reviewed by eLife

Measuring the tolerance of the genetic code to altered codon size

1. Erika Alden DeBenedictis
2. Dieter Söll
3. Kevin M Esvelt

• Curated by eLife

  Evaluation Summary:

  Using a phage-based library generation and selection, the authors generated a suite of 4-base decoding tRNAs with improved efficiency in quadruplet decoding. The data represent an important step toward enhancing protein synthesis with 4-base codons. Overall, the approach to generate many tRNA variants with quadruplet anticodons is intriguing and provides a wealth of valuable information to the field. The results, once some of the reviewer concerns have been addressed, should become foundational for the field of synthetic biology.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Reviewed by eLife

Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

1. Sridevi Challa
2. Keun W Ryu
3. Amy L Whitaker
4. Jonathan C Abshier
5. Cristel V Camacho
6. W Lee Kraus

• Curated by eLife

  Evaluation Summary:

  Challa and Ryu et al. systematically evaluated various combinations of ADP-ribose-binding modules to make sensors detecting poly(ADP-ribose). They developed and tested two indicator designs optimized for analyses in cell culture (dimerization-dependent GFP-based) or intact tissues (split Nano luciferase-based). Overall, with further experimental controls and quantification, this timely set of cell biology probes will be useful to study the biological functions of ADP-ribosylation in cultured cells and whole organisms.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

Reviewed by eLife

Heterogeneity of the GFP fitness landscape and data-driven protein design

1. Louisa Gonzalez Somermeyer
2. Aubin Fleiss
3. Alexander S Mishin
4. Nina G Bozhanova
5. Anna A Igolkina
6. Jens Meiler
7. Maria-Elisenda Alaball Pujol
8. Ekaterina V Putintseva
9. Karen S Sarkisyan
10. Fyodor A Kondrashov

• Curated by eLife

  Evaluation Summary:

  The manuscript dives into how protein structure/function robustness to mutation or polymorphism relates across evolutionary distance. The work indicates that evolutionarily related genes will have different shapes of robustness to variation, and that this will not necessarily track with phylogenetic relationships. The conclusions have potential ramifications for protein engineering, protein structure as well as population genetics and phylogenetics.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by eLife

A scalable pipeline for SARS-CoV-2 replicon construction based on de-novo synthesis

1. Ahmet C. Berkyurek
2. Elian Lee
3. Omer Weissbrod
4. Roni Rasnic
5. Ilaria Falciatori
6. Siyuan Chen
7. Yaniv Erlich

Reviewed by ScreenIT

Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host

1. Francesca D'Angelo
2. Elena Fernández-Fueyo
3. Pierre Simon Garcia
4. Helena Shomar
5. Martin Pelosse
6. Rita Rebelo Manuel
7. Ferhat Büke
8. Siyi Liu
9. Niels van den Broek
10. Nicolas Duraffourg
11. Carol de Ram
12. Martin Pabst
13. Emmanuelle Bouveret
14. Simonetta Gribaldo
15. Béatrice Py
16. Sandrine Ollagnier de Choudens
17. Frédéric Barras
18. Gregory Bokinsky

• Curated by eLife

  Evaluation Summary:

  Three of Nature's life-sustaining processes, respiration, photosynthesis, and nitrogen fixation, all rely on proteins (Fe-S protein) that contain simple inorganic cofactors constructed of Fe and S (Fe-S clusters). Fe-S proteins also participate in a huge and diverse array of metabolic processes. As such there has been considerable interest over the past two decades towards understanding how Fe-S clusters are formed and distributed to their cognate proteins. A related issue, the topic of the present work, is: why is it that many Fe-S proteins from diverse microbial species cannot be heterologously produced in Escherichia coli in active forms? This issue is of considerable interest not only from the perspective of microbial Fe-S proteins but also for heterologous expression of active eukaryotic Fe-S proteins. The study provides insights on the phylogenetic and biosynthetic limitations concerning formation of functional heterologously expressed Fe-S proteins.

  (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

Reviewed by eLife