This is the final Codon Index. But it isn’t farewell. A newsletter will arrive in your inbox next week, albeit with some major changes. The paper roundup will remain. See you then.

Envy is a dangerous emotion. But I couldn’t help feeling it when, last week, I saw an article go viral and thought, “Damn, I should have written that!”

It was a news story, published in New Scientist, about cocaine-producing tobacco plants. And its virality makes sense; drugs and genetic engineering are a lethal combo. The news story has (apparently) garnered 150,000+ views while the research paper, as of this writing, has a measly 2,100 views.

The study itself appeared in JACS on November 14th and is interesting for two reasons. It marks the first time that the cocaine biosynthesis pathway, native to coca leaves, has been ported into another plant. And, more importantly, the study reveals two key enzymes that complete the biosynthetic pathway for cocaine and had been missing from prior work.

Those two enzymes, “conveniently” named CYP81AN15 and MT4, convert 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid into methylecgonone, a precursor to cocaine. With their discovery, we now understand and can rebuild the entire biosynthetic pathway for cocaine. Pharmaceutical companies, surely, are giggling with glee as they make plans to place these enzymes into yeast and other microbes to make ultra-pure cocaine and related compounds.

Producing the drug in tobacco plants will have to suffice for now, though. The addition of just six genes coerced the plants to make 60.5 nanograms of cocaine per milligram of dry weight.

The full biosynthetic pathway, as recreated in tobacco plants.

Read more at the Journal of the American Chemical Society.

Magnet-Controlled Proteins

Nearly twenty years ago, Ed Boyden and Karl Deisseroth — two young neuroscientists at Stanford University — sat down together and wrote out all the ways they could imagine to control a neuron. With paper and pen in hand, they considered using chemicals, sound, and other signals to switch the cells ‘on’ and ‘off’. Each idea, in its turn, was scratched out until only one remained: Light.

That fateful decision would give rise, we now know, to optogenetics, a technique in which pulses of light, emitted from an LED or laser beam, trigger action potentials in living neurons or other excitable cells.

But light can do more than control entire cells; it can also fine-tune the activity of individual proteins. The latter works by splitting each protein into two parts and fusing each half to a light-sensitive magnet protein. When these ‘magnets’ are struck by a photon, Bzzzt!, they find their twin, come together, and reunite the divided protein. I’ve previously written about this concept in my article on ‘light-controlled CRISPR gene editing’.

The same thing can be done now with magnetic fields.

A gene taken from ghost catfish, called the "Electromagnetic Perceptive Gene," or EPG, naturally changes its shape when exposed to a magnetic field. For a new preprint, researchers fused EPG to split halves of a protein and showed, in living cells, that they could use magnetic fields to join the halves together. When the magnetic field is switched off, the protein splits again.

Although this is the first report of such a "magnetogenetic" protein switch, the tool is relatively slow — it takes about 60 seconds for the proteins to join together.

Read more at bioRxiv.

Cells Swim Like Magic

In just the last week, a million-plus people have used ChatGPT to compose poems in the style of Emily Dickinson or dream up Shakespearean insults for that guy at work nobody likes. And then, a few days ago, researchers reported that a technique, called Unclearing Microscopy, enables one to see entire cells with the unaided eye. And then this paper came out. All three of these advances are — and I’m not exaggerating here — reminiscent of magic. All three would seem like blasphemous insults to an Omniscient Creator just a few decades ago.

Ons M'Saad @OnsMSaad1

***See your cells without a microscope*** 👀 Introducing #UnclearingMicroscopy, where cells are expanded 8,000 volumetrically and opaqued with light-scattering molecules of high density to reveal cell microstructure with the naked eye bioRxiv: biorxiv.org/content/10.110…

Of these three advances, the third one — the “new paper” — seems most magical of all, and yet is quite simple; it simply describes how to make cells swim. The new study, which appeared in Science Advances last week, goes like this:

Begin with a ‘minimal’ organism, a cell where non-essential genes have been stripped away to leave, hypothetically (but not quite), the smallest possible genome required to sustain life.

Then, find a swimming pathogen, called Spiroplasma eriocheiris, and copy seven genes from its genome.

Add those seven genes to the minimal cell. (Note: Those genes, in their entirety, are encoded by just 8,400 letters of DNA, or roughly 1/357,000th the length of the human genome.)

Place the engineered cells beneath a microscope. Look how they squirm, twist, and dance in motions reminiscent of the pathogens.

On the right, cells with the parasite’s genes twist and tumble while the un-engineered cells sit still. Just under half of the engineered cells “exhibited morphological change and active movements,” according to the paper, “and 13% had a helical shape and swimming motility.”

But wait, there’s more!

For a second experiment, the researchers wondered: Are all 7 of the parasite genes essential for swimming? Answer: No.

Placing just two genes inside of the bacteria (such as genes #5 and #1, or #5 and #4) were sufficient to turn the bacterial cells into helical-shaped swimmers.

This experiment, to me, reveals the beautiful modularity of life’s machines. And it’s reminiscent of magic.

Read more at Science Advances.

The Index 🔻

A collection of research papers published in the last week.

(↑ = recommended article, * = open access, † = review, comment, etc. )

Basic Research

↑*Reconstitution of a minimal motility system based on Spiroplasma swimming by two bacterial actins in a synthetic minimal bacterium. Kiyama H…Miyata M. Science Advances. Link

*Standardized multi-omics of Earth’s microbiomes reveals microbial and metabolite diversity. Shaffer JP…Earth Microbiome Project 500 Consortium. Nature Microbiology. Link

*Trajectories for the evolution of bacterial CO2-concentrating mechanisms. Flamholz AI…Savage DF. PNAS. Link

*Structures of the eukaryotic ribosome and its translational states in situ. Hoffmann PC…Beck M. Nature Communications. Link

Deep mutational scan of a drug efflux pump reveals its structure–function landscape. Meier G…Seeger MA. Nature Chemical Biology. Link

Biomanufacturing, Metabolic Engineering & Biomaterials

↑Discovery and Engineering of the Cocaine Biosynthetic Pathway. Wang Y-J…Huang S-X. JACS. Link

↑*Total enzymatic synthesis of cis-α-irone from a simple carbon source. Chen X…André I. Nature Communications. Link

This study is a remarkable feat of protein and metabolic engineering. A methyltransferase enzyme, engineered to have >10,000-fold higher activity compared with a wildtype version, was placed into bacterial cells. Those cells produced 90 milligrams of cis-α-irone (used in perfume) per liter.

The experiments in this paper are intimidating: Site-saturation mutagenesis, pooled reactions, and molecular simulations were all used to pinpoint which amino acids should be altered to enhance the specificity, activity, and even gene expression levels of the new enzyme.

Conversion of Mevalonate to Isoprenol Using Light Energy in Escherichia coli without Consuming Sugars for ATP Supply. Sano M…Toya Y. ACS Synthetic Biology. Link

*Development of a microbial protease for composting swine carcasses, optimization of its production and elucidation of its catalytic hydrolysis mechanism. Zhai W…Gao Y. BMC Biotechnology. Link

Modular Engineering Strategy to Redirect Electron Flux into the Electron-Transfer Chain for Enhancing Extracellular Electron Transfer in Shewanella oneidensis. Ding Q…Song H. ACS Synthetic Biology. Link

Modulating DHA-Producing Schizochytrium sp. toward Astaxanthin Biosynthesis via a Seamless Genome Editing System. Liu P-Y…Wan X. ACS Synthetic Biology. Link

Metabolic and Microbial Community Engineering for Four-Carbon Dicarboxylic Acid Production from CO2-Derived Glycogen in the Cyanobacterium Synechocystis sp. PCC6803. Hidese R…Hasunuma T. ACS Synthetic Biology. Link

†Microbial lifelines in bioprocesses: From concept to application. Blöbaum L, Haringa C & Grünberger A. Biotechnology Advances. Link

†Recent advances in research for potential utilization of unexplored lichen metabolites. Kalra R, Conlan XA & Goel M. Biotechnology Advances. Link

†Shikimic acid biosynthesis in microorganisms: Current status and future direction. Sheng Q…Zhang B. Biotechnology Advances. Link

*†Boosting lignan-precursor synthesis in yeast cell factories through co-factor supply optimization. Perrin J…Courdavault V. Frontiers in Bioengineering and Biotechnology. Link

Biosensors

*Characterization of Eight Bacterial Biosensors for Microbial Diagnostic and Therapeutic Applications. Vaaben TH, Vazquez-Uribe R & Sommer MOA. ACS Synthetic Biology. Link

Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors. Mathur D…Medintz IL. ACS Synthetic Biology. Link

Computational Tools & Models

↑*Analysis of the first genetic engineering attribution challenge. Crook OM…Bradshaw WJ. Nature Communications. Link

An unknown DNA sequence appears in the wild. It encodes a gene from a pathogen, virus, or infectious microbe. Its origin is a mystery. Where did it come from? Is it natural or engineered? If the latter, who made it?

Machine learning models can now answer the “who,” at least, with remarkable accuracy. For an open competition, called the Genetic Engineering Attribution Challenge, 300 teams competed to connect a given DNA sequence to its likely source. Each team received the same dataset: nearly 82,000 plasmids from the Addgene repository. Those plasmids were deposited by more than 3,700 different labs. A prize pool offered $30,000 to the winners. The “top-scoring teams dramatically outperformed previous models at identifying the true lab-of-origin of engineered plasmid sequences, including an increase in top-1 and top-10 accuracy of 10 percentage points.” Most, but not all, of the winners used convolutional neural networks.

↑*When synthetic biology fails: a modular framework for modelling genetic stability in engineered cell populations. Ingram D & Stan G-B. bioRxiv (preprint). Link

Computer-Based Design of a Cell Factory for High-Yield Cytidine Production. Han B, Dai Z & Li Z. ACS Synthetic Biology. Link

†Machine learning in bioprocess development: from promise to practice. Helleckes LM…Grünberger A. Trends in Biotechnology. Link

*Genetically personalised organ-specific metabolic models in health and disease. Foguet C…Inouye M. Nature Communications. Link

*Deduction of signaling mechanisms from cellular responses to multiple cues. Saha S…Mugler A. npj Systems Biology and Applications. Link

*GeneFriends: gene co-expression databases and tools for humans and model organisms . Raina P…de Magalhães JP. Nucleic Acids Research. Link

CRISPR, DNA Editing & Regulation

↑*Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo. Tiyaboonchai A…Grompe M. Nature Communications. Link

↑*Decorating chromatin for enhanced genome editing using CRISPR-Cas9. Chen E…Doudna JA. PNAS. Link

Another paper showcases the remarkable adaptability of CRISPR gene editing. Cas9 fused to PRDM9, a protein that methylates histones, increases homology-directed repair three-fold compared with wildtype Cas9. There is no increase in off-target editing and the fusion protein works in multiple cell lines.

*Circular single-stranded DNA is a superior homology-directed repair donor template for efficient genome engineering. Xie K…Wu H. bioRxiv (preprint). Link

*Engineering of efficiency enhanced Cas9 and base editors with improved gene therapy efficacies. Yin S…Li D. Molecular Therapy. Link

*Biochemical characterization of the two novel mgCas12a proteins from the human gut metagenome. Kim HS…Choe S. Scientific Reports. Link

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DNA Sequencing, Synthesis & Assembly

↑*Rapid and Accurate Assembly of Large DNA Assisted by In Vitro Packaging of Bacteriophage. Nozaki S. ACS Synthetic Biology. Link

Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase. Wang G…Chen T. ACS Synthetic Biology. Link

*Nanopore sequencing data analysis using Microsoft Azure cloud computing service. Truong L…De Santis D. PLoS ONE. Link

Genetic Circuits

*Implementing re-configurable biological computation with distributed multicellular consortia. Canadell D…Posas F. Nucleic Acids Research. Link

Medicine & Diagnostics

↑*Treatment of epilepsy using a targeted p38γ kinase gene therapy. Morey N…Ittner LM. Science Advances. Link

Enhancing the activity of a specific gene, called p38γ, reduces behavioral deficits and death in two mouse models of epilepsy. The p38γ protein phosphorylates the tau protein — which normally stabilizes the cytoskeleton of neurons — at amino acid 205, a threonine. A gene encoding p38γ was delivered to mouse neurons via an adeno-associated virus. Two months later, these mice were induced to have seizures. Animals with the gene therapy had significantly higher survival rates.
Seizures were induced using a chemical called pilocarpine. Figure taken from Morey N et al. in Science Advances.

↑*Mannose-coupled AAV2: a second generation AAV vector for increased retinal gene therapy efficiency. Mével M…Adjali O. bioRxiv (preprint). Link

↑*A self-powered ingestible wireless biosensing system for real-time in situ monitoring of gastrointestinal tract metabolites. De la Paz E…Mercier PP. Nature Communications. Link

↑*Lecanemab in Early Alzheimer’s Disease. van Dyck CH…Iwatsubo T. The New England Journal of Medicine. Link

*Engineered Lactococcus lactis secreting Flt3L and OX40 ligand for in situ vaccination-based cancer immunotherapy. Zhu J…Liu B. Nature Communications. Link

*Immunosuppressive biomaterial-based therapeutic vaccine to treat multiple sclerosis via re-establishing immune tolerance. Nguyen TL…Kim J. Nature Communications. Link

*Quadruple gene-engineered natural killer cells enable multi-antigen targeting for durable antitumor activity against multiple myeloma. Cichocki F…Miller JS. Nature Communications. Link

*Rational identification of potent and broad sarbecovirus-neutralizing antibody cocktails from SARS convalescents. Cao Y…Xie XS. Cell Reports. Link

*Gene editing of human iPSCs rescues thrombophilia in hereditary antithrombin deficiency in mice. Tang LV…Hu Y. Science Translational Medicine. Link

*DNA origami–based artificial antigen-presenting cells for adoptive T cell therapy. Sun Y…Pei H. Science Advances. Link

Plants

*†Strategies for genotype-flexible plant transformation. Lee K & Wang K. Current Opinion in Biotechnology. Link

*Genetic engineering of complex feed enzymes into barley seed for direct utilization in animal feedstuff. Peng R-H…Yao Q-H. Plant Biotechnology Journal. Link

*†Plant nitrogen availability and crosstalk with phytohormones signalings and their biotechnology breeding application in crops. Xing J…Wan X. Plant Biotechnology Journal. Link

†Biotechnological approaches for predicting and controlling apple storage disorders. Gapper NE, Bowen JK & Brummell DA. Current Opinion in Biotechnology. Link

Protein & Molecular Engineering

↑*Computation-guided redesign of promoter specificity of a bacterial RNA polymerase. Liu X…Raman S. bioRxiv (preprint). Link

↑*A putative design for electromagnetic activation of split proteins for molecular and cellular manipulation. Grady CJ…Gilad AA. bioRxiv (preprint). Link

*Engineering receptors in the secretory pathway for orthogonal signalling control. Mahameed M…Fussenegger M. Nature Communications. Link

*Evolutionary Inspired Engineering of Megasynthetases. Bozhueyuek KAJ…Bode HB. bioRxiv (preprint). Link

*Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase. King E…Li H. Nature Communications. Link

*A growth selection system for the directed evolution of amine-forming or converting enzymes. Wu S…Bornscheuer UT. Nature Communications. Link

*Spatiotemporal control of necroptotic cell death and plasma membrane recruitment using engineered MLKL domains. Taslimi A…Tucker CL. Cell Death Discovery. Link

*Deep-tissue SWIR imaging using rationally designed small red-shifted near-infrared fluorescent protein. Oliinyk OS…Verkhusha VV. Nature Methods. Link

Tissue & Cell Engineering

*Highly efficient reprogrammable mouse lines with integrated reporters to track the route to pluripotency. Elbaz J…Nagy A. PNAS. Link

Tools, Toolkits & Technology

↑*Monitoring of cell-cell communication and contact history in mammals. Zhang S…Zhou B. Science. Link

A simple concept with brilliant execution, this paper shows that synthetic Notch receptors — a type of protein that sits in a cell’s membrane membrane — can be harnessed to turn on genes when two cells make physical contact. The technology was adapted to record the history of cell-cell contacts across the development of an entire mouse heart. It’s quite striking.

*A regulatory toolkit of arabinose-inducible artificial transcription factors for Gram-negative bacteria. Naseri G…Erhardt M. bioRxiv (preprint). Link

Quantification of extracellular proteins, protein complexes and mRNAs in single cells by proximity sequencing. Vistain L…Tay S. Nature Methods. Link

*Single-virus tracking with quantum dots in live cells. Liu H-Y…Pang D-W. Nature Protocols. Link

*Protein structure determination in human cells by in-cell NMR and a reporter system to optimize protein delivery or transexpression. Gerez JA…Riek R. Communications Biology. Link

*Protein painting reveals pervasive remodeling of conserved proteostasis machinery in response to pharmacological stimuli. Cox D…Hatters DM. npj Systems Biology and Applications. Link

Other Interesting Stuff

*Systematic assessment of the replicability and generalizability of preclinical findings: Impact of protocol harmonization across laboratory sites. Arroyo-Araujo M…Kas MJ. PLOS Biology. Link

*A three-photon head-mounted microscope for imaging all layers of visual cortex in freely moving mice. Klioutchnikov A…Kerr JND. Nature Methods. Link

*Genomic analysis of sewage from 101 countries reveals global landscape of antimicrobial resistance. Munk P…Aarestrup FM. Nature Communications. Link

*When did mammoths go extinct? Miller JH & Simpson C. Nature. Link

And the response: Link

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Cocaine from Tobacco Plants: Index #77