The modern genome represents the culmination of 3.8 billion years of the evolution of life on Earth and encodes the astonishingly complex information we observe in the biosphere. Despite more than 70 years of molecular biology research, we still don’t understand exactly how this complex information is encoded in the genome. For example, in the E. coli genome, only 48.9% of the genes have been functionally identified, while in the Saccharomyces cerevisiae genome, more than 1,000 of the approximately 6,000 genes have unknown functions.
Synthetic biology is a young interdisciplinary field that combines biology with cutting-edge engineering to benefit the agriculture, manufacturing, fuel, environment and medical sectors. With advances in synthetic biology, complex heterologous pathways have been engineered for a wide range of applications, including production of proliferative molecules, utilization of inexpensive nutrient sources, and detection of pollutants or diseases. However, the complexity of biological systems hinders our ability to modify existing genomes, which can lead to genetic incompatibility, destabilization of heterologous pathways, and low yields of products due to competition for cellular resources.
As whole genome synthesis becomes achievable and less expensive, one solution is to unlock a more complete understanding of biology at the genome level by constructing minimal genomes. Synthesizing minimal genomes will also provide insights into the extent of genome fragmentation and remodeling, the role of non-coding DNA and repetitive elements, and the extent to which biological epigenetic regulation can be engineered through genome redesign. Furthermore, due to improved stability, increased predictability through modeling, and greater biosynthetic capacity, they could serve as simplified and superior cellular bases for biotechnological applications (Fig. 1).
Figure 1 | Applications of Synthetic and Synthetic Minimal Genomes
On April 8, 2023, the Thomas C. Williams team of the ARC Center of Excellence for Synthetic Biology published an article titled “Trimming the genomic fat: minimizing and functionalizing genomes using synthetic biology” on Nature Communications, reviewing the past and present research on genomics. The work done on minimization and refunctionalization highlights recent advances facilitated by synthetic genomics and provides a critical assessment of its potential for industrial applications.
Synthetic Genomics Enables New Possibilities for Genome Minimization
With the reduction in the cost of DNA synthesis and the development of large-scale DNA assembly technologies, “bottom-up” genome minimization and refunctionalization can now be achieved through whole-genome design and synthesis. These “bottom-up” approaches rely on the de novo synthesis of new genomes, or the gradual replacement of existing genomes with rationally designed and chemically synthesized DNA.
／Synthesis of Mycoplasma Genome／
In 2002, poliovirus cDNA (~7.5 kb) was the first to be chemically synthesized. Following the successful synthesis of the poliovirus genome, Gibson et al. synthesized the first prokaryotic genome, the 582,970 base-pair genome of Mycoplasma genitalium, a bacterium with the smallest genome grown in pure culture. Since the growth rate of M. genitalium is very slow, two types of Mycoplasma with faster growth rate were selected for follow-up study. Mycobacteria were selected for de novo genome synthesis and M. carinii was selected as the recipient cell for the synthesis of the mycobacterial genome.
Gibson et al. successfully completed a synthetic genome of mycobacteria (JCVI-syn1.0) in 2010, which contained four marker sequences designed to distinguish the synthetic genome from the wild-type genome. A 1.08 Mb genome was assembled from 1078 overlapping 1 kb DNA fragments in yeast by transformation and homologous recombination, and then transplanted into M. capricolum recipient cells to create a new synthetic strain exhibiting a similar expression to the fungus Bacillus type (Fig. 2a).
Figure 2 | Construction of a synthetic genome
／Synthesis of a minimal mycobacterial genome ／
In a subsequent study, the team applied whole-genome design and synthesis to minimize mycobacterial genomes (Fig. 3). The minimal genome was originally designed based on existing transposon mutagenesis data and molecular biology knowledge. However, the original design did not result in a viable strain. Subsequently, a Tn5 mutagenesis study was performed to identify essential, non-essential and quasi-essential genes. A viable minimal genome (JCVI-syn2.0) was obtained by retaining quasi-essential genes and avoiding deletion of synthetic lethal pairs.
Furthermore, another round of Tn5 mutations in syn2.0 resulted in the deletion of an additional 42 genes, resulting in an approximately minimal genome (JCVII-syn3.0) with a total deletion of 428 genes. The genome of the syn3.0 strain is 531 kb, which is smaller than any autonomously replicating cell known in nature. Compared with syn1.0, the genome is reduced by 51%, and the doubling time is about 180 min, which is slower than The doubling time of the latter is about 60 minutes. However, it grows much faster than the 16-hour doubling time of M. genitalium. It can be inferred that there is a balance between removing as many genes as possible and maintaining a certain level of growth fitness.
Figure 3 | “Bottom-up” construction of the minimal Mycobacterium mycoplasma genome (JCVI-syn3.0)-T
／ Synthesis of Recombinant Escherichia coli Genome ／
Advances in synthetic genomics have also facilitated the reassignment of biological codons. Total synthesis was performed in E. coli to remove three codons genome-wide, resulting in a synthetic E. coli genome with 61 codons. In the synthetic E. coli genome, two serine codons and a stop codon were replaced, and an essential tRNA gene was deleted.
The method uses CRISPR-Cas9 and lambda-mediated recombination to replace genomic DNA with recombinant DNA from BACs. By integrating four to five fragments of approximately 100 kb, seven strains with partially synthetic genomes were generated , with step-by-step genome exchange synthesis (genes) achieved by alternating successive REXER cycles using positive and negative selection (Fig. 2b). Finally, through coupled transfer and recombination, the large coding fragments were combined into a complete synthetic coding genome, called “Syn61”.
The resulting codon-squeezing strain Syn61 offers great potential for the production of proteins with novel functions by codon reassignment as well as for industrial bioprocessing, as they resist phage contamination through genetic code incompatibility.
However, releasing more codons is very challenging because large-scale genome recoding will not only increase the technical difficulty of DNA synthesis and assembly, but also affect GC content, protein expression and global epigenetic signaling, which may lead to severe Fitness deficit or lethality. In 2016, Ostrov et al. reported progress in the design, synthesis, and testing of the codon E. coli genome. They have verified the function of 63% of the coding genes, and they are still assembling fully coded strains.
／Synthesis of Saccharomyces cerevisiae genome ／
Parallel to the success of de novo bacterial genome synthesis, a global consortium led by NYU’s Jeff Bock has been working on an ambitious Sc2.0 project to construct the first synthetic eukaryotic genome, the vinculum Yeast genome. The goal of the project is not only to gain insight into yeast genomics, but also to create a simpler yeast cell with fitness comparable to wild type that can be downsized and reconfigured for different engineering purposes.
The following changes were made in the design of Sc2.0: destabilizing or redundant elements including retrotransposons, subtelomeric repeats, and introns were removed; repetitive transfer RNA (tRNA) genes were relocated to On the “new chromosome”, its function and stability are tested separately; TAG stop codons are exchanged with TAA for future codon reassignment; native telomeres are replaced by standardized synthetic versions; codon strings are recoded to the same Sense codons are used as “PCR tags”, which can be used as markers to distinguish synthetic sequences from wild-type sequences. Initially, construction of synthetic chromosomes began with oligonucleotide assembly of 750 bp building blocks, which were then assembled in yeast to generate microblocks (Fig. 2c).
Nine strains containing one synthetic chromosome were reported to grow comparable to wild-type strains. A global team is building a fully synthetic yeast genome. Once complete, the synthetic genome will reduce genome size by nearly 8% and will serve as a genome-wide diversification and minimization platform.
“Bottom-up” genome construction enables novel design changes at the genome-wide level. However, genome-scale synthesis is currently still prohibitively expensive, especially for eukaryotic genomes. There will also be some hard-to-composite areas that will need to be recoded.
As more knowledge about genome biology and gene regulation is gained through the study and rewriting of genomes, the problem of our inability to design minimally functional genomes from scratch continues to emerge. However, the iterative design, build, and test required to continue working on minimal genomes will ultimately refine our understanding and capabilities in genome design.