Synthetic biology offers the opportunity to build solutions for improved capture and conversion of carbon dioxide (CO₂) that outcompete those evolved by nature. Here we demonstrate the design and construction of a new-to-nature CO₂-fixation pathway, the reductive tricarboxylic acid branch/4-hydroxybutyryl-CoA/ethylmalonyl-CoA/acetyl-CoA (THETA) cycle. The THETA cycle encompasses 17 enzymes from 9 organisms and revolves around two of the most efficient CO₂-fixing enzymes described in nature, crotonyl-CoA carboxylase/reductase and phosphoenolpyruvate carboxylase. Here using rational and machine learning-guided optimization approaches, we improved the yield of the cycle by two orders of magnitude and demonstrated the formation of different biochemical building blocks directly from CO₂. Furthermore, we separated the THETA cycle into three modules that we successfully implemented in vivo by exploiting the natural plasticity of Escherichia coli metabolism. Growth-based selection and/or ¹³C-labelling confirmed the activity of three different modules, demonstrating the first step towards realizing highly orthogonal and complex CO₂-fixation pathways in the background of living cells.

Fractals are patterns that are self-similar across multiple length-scales¹. Macroscopic fractals are common in nature^2–4; however, so far, molecular assembly into fractals is restricted to synthetic systems^5–12. Here we report the discovery of a natural protein, citrate synthase from the cyanobacterium Synechococcus elongatus, which self-assembles into Sierpiński triangles. Using cryo-electron microscopy, we reveal how the fractal assembles from a hexameric building block. Although different stimuli modulate the formation of fractal complexes and these complexes can regulate the enzymatic activity of citrate synthase in vitro, the fractal may not serve a physiological function in vivo. We use ancestral sequence reconstruction to retrace how the citrate synthase fractal evolved from non-fractal precursors, and the results suggest it may have emerged as a harmless evolutionary accident. Our findings expand the space of possible protein complexes and demonstrate that intricate and regulatable assemblies can evolve in a single substitution.

The field of genetic engineering was born in 1973 with the “construction of biologically functional bacterial plasmids in vitro”. Since then, a vast number of technologies have been developed allowing large-scale reading and writing of DNA, as well as tools for complex modifications and alterations of the genetic code. Natural genomes can be seen as software version 1.0; synthetic genomics aims to rewrite this software with “build to understand” and “build to apply” philosophies. One of the predominant model organisms is the baker’s yeast Saccharomyces cerevisiae. Its importance ranges from ancient biotechnologies such as baking and brewing, to high-end valuable compound synthesis on industrial scales. This tiny sugar fungus contributed greatly to enabling humankind to reach its current development status. This review discusses recent developments in the field of genetic engineering for budding yeast S. cerevisiae, and its application in biotechnology. The article highlights advances from Sc1.0 to the developments in synthetic genomics paving the way towards Sc2.0. With the synthetic genome of Sc2.0 nearing completion, the article also aims to propose perspectives for potential Sc3.0 and subsequent versions as well as its implications for basic and applied research.

Since the first synthetic gene was synthesized in 1970s, the efficiency and the capacity of made-to-order DNA sequence synthesis has increased by several orders of magnitude. Advances in DNA synthesis and assembly over the past years has resulted in a steep drop in price for custom made DNA. Similar effects were observed in DNA sequencing technologies which underpin DNA-reading projects. Today, synthetic DNA sequences with more than 10000bps and turn-around times of a few weeks are commercially available. This enables researchers to perform large-scale projects to write synthetic chromosomes and characterize their functionalities in vivo. Synthetic genomics opens up new paradigms to study the genome fundamentals and engineer novel biological functions.

Microbial diversity is magnificent and essential to almost all life on Earth. Microbes are an essential part of every human, allowing us to utilize otherwise inaccessible resources. It is no surprise that humans started, initially unconsciously, domesticating microbes for food production: one may call this microbial domestication 1.0. Sourdough bread is just one of the miracles performed by microbial fermentation, allowing extraction of more nutrients from flour and at the same time creating a fluffy and delicious loaf. There are a broad range of products the production of which requires fermentation such as chocolate, cheese, coffee and vinegar. Eventually, with the rise of microscopy, humans became aware of microbial life. Today our knowledge and technological advances allow us to genetically engineer microbes - one may call this microbial domestication 2.0. Synthetic biology and microbial chassis adaptation allow us to tackle current and future food challenges. One of the most apparent challenges is the limited space on Earth available for agriculture and its major tolls on the environment through use of pesticides and the replacement of ecosystems with monocultures. Further challenges include transport and packaging, exacerbated by the 24/7 on-demand mentality of many customers. Synthetic biology already tackles multiple food challenges and will be able to tackle many future food challenges. In this perspective article, we highlight recent microbial synthetic biology research to address future food challenges. We further give a perspective on how synthetic biology tools may teach old microbes new tricks, and what standardized microbial domestication could look like.

Background: Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction.Results: We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified.Conclusions: We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.