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Cost-effective, environmentally sustainable and energy-efficient ways to address rising atmospheric CO₂ levels are urgently needed. Here the authors combine electrochemical reduction of CO₂ to formate with biosynthetic conversion of formate to the universal building block acetyl-CoA using a synthetic metabolic pathway called ReForm.

Allosteric transcription factors (aTFs) are promising tools for environmental and human health monitoring. Here the authors develop a multi-objective, machine learning-guided method to engineer an aTF-based portable diagnostic for environment sensing of lead in drinking water at the legal limit.

The GlycoSCORES method, which involves cell-free protein expression and substrate-site profiling of glycosyltransferase enzymes by SAMDI–MS, enables the identification of glycosylation tags for glycoengineering efforts.

The inability to produce recombinant phosphoproteins has hindered research into their structure and function. Here the authors develop a cell-free protein synthesis platform to site-specifically incorporate phosphoserine into proteins at high yields, and recapitulate a MEK1 kinase signalling cascade.

As part of the first anniversary issue of Nature Chemical Engineering, we present a collection of opinions from 40 researchers within the field on what they think are the most exciting opportunities that lie ahead for their respective topics.

Cell-free protein synthesis allows for producing proteins without the need of a host organism, thus sparing the researcher experimental hassle. Here, the authors developed a cell-free synthesis method that enables incorporating non-standard amino acids in the product.

Automated 3D design produces rapid and near-atomically accurate predictions of RNA tertiary structure as well as the ability to generate complex RNA machines such as functional single-stranded tethered ribosomes, and enhancement of the binding properties of small-molecule RNA aptamers.

Ribosomes have evolved to polymerize L-α-amino acids into proteins comprising a peptide backbone. Here, a pyridazinone backbone is formed using ribosomes in vitro, producing a variety of sequence-defined alternating block-copolymers.

An attractive route for carbon-negative synthesis of biochemical products is the reverse β-oxidation pathway coupled to the Wood-Ljungdahl pathway. Here the authors use a high-throughput in vitro prototyping workflow to screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity.

Glycosylation of human IgG antibodies in bacterial hosts has proven challenging. Here, the authors identify a bacterial enzyme enabling E. coli to glycosylate IgGs at native sites, thereby advancing therapeutic antibody development in this host.

Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins. Here, the authors develop a high-throughput workflow combining cell-free gene expression with AlphaLISA to rapidly characterize and engineer PTMs on both proteins and peptides.

The iPROBE platform accelerates the design and optimization of engineered biosynthetic pathways using a combination of cell-free protein synthesis, in vitro pathway assembly and a scoring system to identify high-performing combinations.

Directed evolution of the ribosome is challenging because the requirement of cell viability limits the mutations that can be made. Here the authors develop a platform for in vitro ribosome synthesis and evolution (RISE) to overcome these constraints.

Flexizymes have been used to expand the scope of chemical substrates for ribosome-directed polymerization in vitro. Here the authors deduce design rules of Flexizyme-mediated tRNA acylation that more effectively predict the incorporation of new monomers into peptides.

Two industrial chemicals are sustainably produced at large scale by microbial gas fermentation.

While the ribosome has been harnessed for synthetic biology, designing ribosomes has remained challenging. Here, the authors demonstrate a community science approach for rational design of ribosomes with beneficial properties.

Evolink, a method developed to co-evolve structurally adjacent but sequence-distant sites in molecular machines, was used in concert with computational modeling to evolve an improved tethered ribosome variant.

While machine learning shows promise in expanding protein engineering efforts, its potential is limited by the challenge of gathering large datasets of sequence-function relationships. Here, authors introduce a platform that integrates cell-free DNA assembly and gene expression to accelerate enzyme engineering.

Dynamic control over protein function is a central challenge in synthetic biology. Here the authors present an integrated computational and experimental workflow for engineering reversible protein switches; metal-chelating unnatural amino acids genetically encoded into two conformationally dynamic enzymes to yield robust switches.

Backbone extended monomers are poorly compatible with the natural ribosomes, impeding their polymerization into polypeptides. Here the authors design non-canonical amino acid analogs with cyclic structures or extended carbon chains and used an engineered ribosome to improve tRNA-charging and incorporation into peptides.

Constructing biosynthetic pathways to study and engineer glycoprotein structures is difficult. Here, the authors use GlycoPRIME, a cell-free workflow for mixing-and-matching glycosylation enzymes, to evaluate 37 putative glycosylation pathways and discover routes to 18 new glycoprotein structures

Cell-free systems enable the design of biosynthetic pathways for sustainable chemical synthesis. Here the authors create an integrated framework for accelerating design using extracts from genetically rewired strains.

Synthetic biology is defined by Design-Build-Test-Learn cycles. Recent advances in machine learning are changing the landscape; thus, we propose that “Learning” can precede “Design”. Moreover, adopting cell-free platforms can further accelerate “Building” and “Testing” for megascale data generation and models.

Ribo-T is a tethered ribosome complex capable of orthogonal ribosome-mRNA functionality, but has low activity. Here the authors evolve new tether designs that support faster growth and increased protein expression.

Michael Talkowski and colleagues analyze balanced chromosomal abnormalities in 273 individuals by whole-genome sequencing. Their findings suggest that sequence-level resolution improves prediction of clinical outcomes for balanced rearrangements and provides insight into pathogenic mechanisms such as altered gene regulation due to changes in chromosome topology.

Cell-free gene expression systems are an attractive platform for biomanufacturing and synthetic biology. Here the authors characterize native membrane vesicles in E. coli extracts for improved glycoengineering.

Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists. Here the authors present the CRISPRkit, a cost-effective educational tool that enables high school students to perform CRISPR experiments affordably and safely without prior experience, using smartphone-based quantification and an automated algorithm for data analysis.

The ability to produce homogeneous glycoproteins is expected to advance fundamental understanding in glycoscience, but current in vivo-based production systems have several limitations. Here, the authors develop an E. coli extract-based one-pot system for customized production of N-linked glycoproteins.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with potent biological activities but a vast majority of RiPP gene clusters remain unexplored in microbial genomes. Here, the authors report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs.

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen specific hits. Here, the authors build an antibody screening workflow leveraging cell-free protein synthesis that enables expression and evaluation of hundreds of antibody fragments in less than 24 h.

Expanding the range of amino acids polymerizable by ribosomes could enable new functionalities to be added to polypeptides. Now, the genetic code has been reprogrammed using a reconstituted in vitro translation system to enable synthesis of unnatural peptides with unmatched flexibility.

Morphology of metabolosomes affects the encapsulated pathway performance. Here, the authors combine experimental characterizations with structural and kinetic modeling to reveal how the shell protein PduN changes the morphology of 1,2-propanediol utilization (Pdu) metabolosome and how this morphology shift impacts Pdu function.

Ribosome engineering is an emerging powerful approach for synthetic protein synthesis. Here the authors invert the Ribo-T system, using the engineered ribosome to translate the proteome while the native ribosome translates specific mRNA.

Multi-ancestry genome-wide association analyses identify new risk loci for Parkinson’s disease, and fine-mapping and co-localization analyses implicate candidate genes whose expression is associated with disease susceptibility.

The tethered ribosome system Ribo-T supports cell proliferation though at a reduced rate. Here the authors show this is due to slower ribosome assembly instead of reduced functionality.

Access to glycoenzymes for basic and applied research is limited by difficulties with their recombinant expression. Here, the authors describe a universal strategy for converting membrane-bound glycosyltransferases into water-soluble biocatalysts, which are expressed at high levels with retention of activity.

In this Review, Zuniga and colleagues discuss partial orchiectomy as a viable alternative to radical surgery for selected patients with testicular lesions. Although currently only suitable for men with bilateral germ cell tumors or a solitary testicle, organ-sparing techniques might soon be considered appropriate for men with a normal contralateral testis.

Genome-wide association analysis of gout and urate identifies 148 new loci, implicating biological pathways and prioritizing candidate genes involved in inflammatory processes.

Information about O-glycosylation site regulation and occupancy in the human proteome is limited. Here, the authors identify GalNAc transferase-specific glycan sites in human keratinocytes and describe their occupancy.

Non-standard amino acids are incorporated into proteins at large numbers of sites using evolved translation components in recoded bacteria.

Primary open-angle glaucoma (POAG) is highly heritable, yet not well understood from a genetic perspective. Here, the authors perform a meta-analysis of genome-wide association studies in 34,179 POAG cases, identifying 44 previously unreported risk loci and mapping effects across multiple ethnicities.

Using new letters of DNA to encode information promises to expand the genetic code in a transformative way. A new semisynthetic organism has been created that uses 67 codons for protein biosynthesis, with three new codons based on unnatural nucleotides.

An orthogonal O-glycan biosynthesis system was engineered in Escherichia coli to support the production of glycoproteins displaying human mucin O-glycans, including Tn antigens, in living bacteria and in cell-free extracts.

Inspired by nature, a synthetic carbon fixation cycle builds complex molecules directly from CO₂. Building metabolism from the ground up requires several innovative advancements — now, a strategy to balance carbon demands in a complex metabolic network is explored.