Researchers Complete Synthetic Yeast Chromosome, Advancing Genome Engineering

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMJan 29 2025

Synthetic genomics has emerged as a transformative field, enabling the design and construction of artificial genomes to explore biological systems and enhance biomanufacturing capabilities.

In a recent study published in Nature Communications, a team of researchers constructed a fully synthetic chromosome, named synXVI, as part of the Sc2.0 project — a global initiative to redesign the genome of the yeast Saccharomyces cerevisiae.

The study described the iterative design and debugging performed on the 902,994 base pairs of synXVI to overcome growth defects and optimize functionality.

Study: Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome. Image Credit: vectorfusionart/Shutterstock.com

Synthetic Chromosomes

Advances in synthetic biology have paved the way for constructing synthetic genomes to investigate genetic functions and optimize biological systems.

Synthetic chromosomes allow researchers to explore large-scale genomic modifications, offer insights into cellular processes, and enhance the production of critical biomolecules. Previous applications of synthetic genomics have predominantly involved bacteria and viruses.

The Sc2.0 project represents the first attempt to design and synthesize a eukaryotic genome, using Saccharomyces cerevisiae as a model organism. This species of yeast is widely used in research and industry due to its genetic malleability and metabolic versatility.

Furthermore, a key feature of Sc2.0 is the introduction of loxPsym or symmetrical lox sites that enable targeted genomic rearrangements.

However, challenges such as growth defects and genomic instability have been observed in synthetic strains, which have been attributed to unintended disruptions in essential genomic regions.

Addressing these challenges is critical for improving the design and implementation of synthetic genomes, especially for the future applications of this technology in complex eukaryotic systems.

The Current Study

The study involved the design, construction, and debugging of synXVI, a synthetic chromosome of Saccharomyces cerevisiae, using a multi-phase approach.

Initially, the chromosome was synthesized in segments called megachunks, which were then cloned into vectors and integrated into yeast strains through homologous recombination. Each megachunk contained overlapping regions for seamless integration.

To identify and resolve functional defects, the researchers employed the Clustered Regularly Interspaced Short Palindromic Repeats Design and Debugging Using Genetic Screens or CRISPR-D-BUGS protocol, which uses targeted genomic modifications to pinpoint defective loci.

They performed whole-genome sequencing and transcriptional assays to identify growth-inhibiting mutations caused by inserted loxPsym sites in the 5' untranslated regions (UTRs) of essential genes. The iterative debugging included restoring native sequences for defective genes such as CTR1 (a copper transporter) and GIP3 (involved in cell division), which were disrupted by loxPsym insertions.

Additionally, a transfer ribonucleic acid (tRNA) neochromosome carrying 17 relocated tRNA genes was introduced to compensate for their removal from synXVI and to address the growth defects linked to tRNA insufficiency.

The study then used numerous fitness assays, including spot dilution tests, to evaluate strain performance under various conditions, such as growth on glycerol-based media and elevated temperatures.

In parallel, the researchers also redesigned synXVI in silico to optimize it to prevent future disruptions. These adjustments were also aimed at minimizing unintended interactions between synthetic elements and cellular machinery.

Results

The study found that the initial design of synXVI caused growth defects in yeast due to unintended disruptions in essential genetic elements. Specifically, the insertion of loxPsym sites in the 5' UTRs of key genes, such as CTR1 and GIP3, interfered with gene expression, leading to poor growth on non-fermentable carbon sources and under high-temperature conditions.

The results showed that restoration of native sequences for these genes improved cellular fitness, and demonstrated the critical role of proper regulatory sequences in synthetic genomes.

Additionally, the removal of 17 tRNA genes from synXVI had previously contributed to growth defects. Therefore, the introduction of a synthetic tRNA neochromosome that carried these missing tRNA genes significantly enhanced the growth, particularly under stress conditions. This also highlighted the importance of maintaining tRNA redundancy in genome-scale modifications.

The analysis involving CRISPR-D-BUGS also identified additional defective loci, including disruptions in mitochondrial ribosomal protein genes such as MRPL51, which affected mitochondrial function. Complementing these genes with native sequences further improved growth performance.

Furthermore, the in silico redesign of synXVI resulted in a refined version with various optimizations, including the strategic placement of loxPsym sites. These changes minimized the likelihood of disrupting essential genetic elements and improved the overall stability and functionality of the synthetic chromosome.

Conclusions

In summary, the team successfully constructed and optimized a synthetic chromosome called synXVI in yeast by addressing problems such as growth defects through iterative debugging and redesign.

The major improvements included restoring disrupted regulatory sequences, reintroducing essential tRNA genes, and refining genomic architecture.

These advancements highlighted the importance of comprehensive design-build-test cycles in synthetic genomics. Moreover, these findings also provide a foundation for future applications, including the development of synthetic genomes for complex organisms and industrially relevant systems.