Biology subjects

Schindler, D.

Publications and source records attributed to Schindler, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Coupled enzyme discovery, evolution and synthetic yeast chassis adaptation for microbial biopolymer valorisation

The valorisation of biological polymers requires microbial systems that can both access recalcitrant substrates and convert the resulting carbon into useful products. Although microbial genome and metagenome resources provide an expanding reservoir of candidate depolymerizing and modifying enzymes, most discovery workflows remain disconnected from enzyme optimisation and host adaptation. Here we present a coupled sequence-based enzyme discovery, enzyme evolution and synthetic yeast chassis adaptation strategy for microbial biopolymer valorisation. Focusing on laccases for the depolymerisation of lignin as a proof of concept, we combine sequence data mining for enzyme discovery, modular yeast surface display for functional screening, directed evolution for enzyme optimisation and synthetic yeast genome diversification for chassis improvement. In our study, surface display enabled functional benchmarking and recovery of improved laccase variants and synthetic-genome-enabled diversification provided a route to explore host configurations that influence display and enzyme performance. By integrating enzyme-level and chassis-level optimisation, this framework addresses a central bottleneck in converting microbial biodiversity by computational sequence repository mining into deployable biomanufacturing systems. Our results establish laccases as tractable entry points for oxidative biopolymer conversion and provide a generalizable platform for engineering yeast systems for sustainable carbon valorisation.

synthetic biology↗

bioRxiv2025

Drift, selection and convergence in the evolution of a nonribosomal peptide

Nonribosomal peptides play important roles in microbial ecology and medicine, for example as toxins and antibiotics such as penicillin1. Like ordinary proteins, nonribosomal peptides are subject to evolutionary change, which is driven by rampant recombinations of their synthetases2. This makes it difficult to study the evolutionary histories of nonribosomal peptides, because recombination is a serious challenge for phylogenetic inference3. Here we use recombination-aware phylogenetics and ancestral sequence reconstruction to retrace the evolution of GameXPeptides, nonribosomal peptides entomopathogenic bacteria use to kill their insect prey4. By untangling the complex histories of their synthetases, we discover dozens of new GameXPeptides and show striking patterns of structural convergence. In analogy to classic statistical test for natural selection acting on nucleotide mutations, we develop a similar test for whether selection acts to fix recombinations. We provide statistical and experimental evidence of both neutral genetic drift and natural selection in the diversification of GameXPeptides. GameXPeptides variants rapidly evolved highly insect specific toxicities, suggesting that their diversification may be driven by an evolutionary arms race. Our work suggests that small, fast evolving alteration to nonribosomal peptides can overcome host resistance on short timescales, with important implications for the evolution of resistance evading antibiotics.

evolutionary biology↗

bioRxiv2025

Machine learning applied to otolith microchemical data to discriminate stock of origin in salmon

A key challenge in ecosystem-based fisheries management is sustainably harvesting co-occurring species and populations that differ in their vulnerability to fishing. This challenge is exemplified in western Alaska Chinook salmon, where recent population declines have led to closures of in-river subsistence fisheries, but identifying the cause of these declines is limited by an inability to identify the river of origin for marine-caught fish in offshore fisheries that target more abundant species and populations. This problem is particularly acute for estimating the impacts of bycatch on individual salmon populations which have demographic independence but no genetic differentiation from which stock assignments can be made. Here we use machine learning approaches to assess the efficacy of using the microchemical history preserved in fish otoliths to assign individuals to their river of origin in western Alaska. We tested the classification ability of three machine learning algorithms (Random Forest, K-Nearest Neighbors, and Support Vector Machines) combined with two time series smoothing techniques (Moving Average and Generalized Additive Models) to classify Chinook salmon to their river of origin using otolith time series data. Model accuracy ranged from 71.9% to 92.5%, with optimal performance achieved by Random Forest applied to GAM-smoothed data. Watershed-specific performance ranged from 86.8% to 93.7%, with most misclassifications occurring between spatially proximate Kuskokwim and Nushagak rivers. Raw predicted probabilities from classification algorithms were calibrated to reflect true class probabilities, enabling the incorporation of model results into decision analyses with explicit consideration of misclassification risk tolerance. The success of these models offers immediate utility for estimating marine mortality impacts across the regions three major river systems as well as an opportunity to understand commercial fisheries impacts on individual populations at substantially finer spatial scales than had been previously possible.

ecology↗

bioRxiv2025

High-throughput engineering and modification of non-ribosomal peptide synthetases based on Golden Gate assembly

Non-ribosomal peptide synthetases (NRPS) are multimodular enzymes that produce complex peptides with diverse biological activities, potentially being used as clinical drugs. However, the pharmaceutical applications of such natural peptides often require further derivatisation and modification of the peptide backbone, mainly performed by chemical synthesis. A sustainable alternative resembles the in vivo engineering of NRPS to change and modify the enzyme properties rationally and, thus, the produced products. The novel NRPS engineering concept, the eXchange Unit Thiolation domain (XUT), allows the efficient modular assembly of different natural NRPS fragments to form hybrid NRPS that produce defined peptides. In this study, we describe a Golden Gate assembly (GGA) method for efficient high-throughput generation of novel and engineered NRPS libraries utilising the XUT concept. This method was applied to generate over 100 novel NRPS with the possibility of changing starter, elongation, and termination modules, respectively. Additionally, we applied this method for targeted modification of the xenoamicin biosynthetic gene cluster (BGC) XabABCD from Xenorhabdus doucetiae, resulting in the generation of 25 novel xenoamicin derivatives. Graphical AbstractA Golden Gate assembly (GGA) method was developed for the efficient assembly of natural and engineered non-ribosomal peptide synthetases (NRPS). This method has enabled the creation of NRPS libraries to generate novel peptides in high-throughput as well as the targeted derivatisation of natural products (NP). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/650154v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1161805org.highwire.dtl.DTLVardef@1832a43org.highwire.dtl.DTLVardef@4bc6b8org.highwire.dtl.DTLVardef@e3908d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Specificities of Chemosensory Receptors in the Human Gut Microbiota

The human gut is rich in metabolites and harbors a complex microbial community, yet the sensory repertoire of its commensal bacteria remains largely uncharacterized. Here we systematically mapped ligand specificities of extracytoplasmic sensory domains from twenty members of the human gut microbiota, with a primary focus on the abundant and physiologically important class of Clostridia. We identified diverse metabolites as specific stimuli for three major functional classes of transmembrane receptors. We further characterized novel subsets of sensors belonging to the Cache superfamily, specific for lactate, dicarboxylic acids, and for uracil and short-chain fatty acids (SCFAs), respectively, and investigated the evolution of their ligand specificity. Structural and biochemical analysis of the newly described dCache_1UR domain revealed an independent binding of uracil and SCFA at distinct modules. Altogether, we could identify or predict specificities for over a half of the Cache-type chemotactic sensors in the selected gut commensals, with the carboxylic acids representing the largest class of ligands. Among those, the most commonly found specificities were for lactate and formate, indicating particular importance of these metabolites in the human gut microbiome and consistent with their observed beneficial impact on the growth of selected bacterial species.

microbiology↗

bioRxiv2024

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

evolutionary biology↗

bioRxiv2024

In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly

Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three nucleotide overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we term In-Cloning. In-Cloning is downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnets group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow called Out-Cloning to rapidly generate Golden Gate acceptor plasmids. Out-Cloning uses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.

synthetic biology↗

bioRxiv2024

A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs

All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying the adaptation to such changes. sRNAs base-pair with their target mRNAs, allowing rapid modulation of the proteome. This post-transcriptional regulation is usually facilitated by RNA chaperones, such as Hfq. sRNAs have a potential as synthetic regulators that can be modulated by rational design. In this study, we use a library-based approach and an oxacillin susceptibility assays to investigate the importance of the seed region length for synthetic sRNAs based on RybB and SgrS scaffolds in Escherichia coli. In the presence of Hfq we show that 12 nucleotides are sufficient for regulation. Furthermore, we observe a scaffold-specific Hfq-dependency and processing by RNase E. Our results provide information for design considerations of synthetic sRNAs in basic and applied research.

synthetic biology↗

bioRxiv2024

T4 phage RNA is NAD-capped and alters the NAD-capepitranscriptome of Escherichia coli during infection through a phage-encoded decapping enzyme

Nicotinamide adenine dinucleotide (NAD+) serves as a cap-like structure on cellular RNAs (NAD-RNAs) across all domains of life, including Escherichia coli. Beyond its role in metabolism, NAD+ also functions as a regulatory molecule in phage defense mechanisms. However, NAD-RNAs have not yet been identified during bacteriophage infections, and the mechanisms governing their synthesis and degradation in this context remain unknown. To address this gap, we used T4 phage infection of E. coli as a defined and well-characterized model system to study NAD-RNAs at the virus-host interface. Here, we report the first identification and characterization of NAD-RNAs during phage infection. Using time-resolved NAD captureSeq, we identified NAD-capped host and T4 phage transcripts and observed that the set of enriched NAD-capped RNAs varies across different infection phases. Importantly, NAD captureSeq identifies NAD-capped transcripts based on enrichment and does not directly report the fraction of molecules that are NAD-capped. Consequently, temporal changes in enrichment may reflect altered NAD-capping, altered transcript abundance, or a combination of both. We provide evidence that NAD-RNAs are generated by the host RNA polymerase by initiating transcription with NAD+ at canonical transcription start sites. By quantifying intracellular NAD+ and bulk NAD-capped RNA during infection, we observe parallel decreases in both parameters over the course of infection. Furthermore, we characterize NudE.1, a T4 phage-encoded Nudix hydrolase previously shown to have in vitro NAD-RNA decapping activity. Together, our work presents the first time-resolved analysis of an RNA modification in a host-phage system, defining the landscape, dynamics, and turnover of NAD-capped RNAs during infection and providing a framework for future studies addressing their regulatory functions in phage biology.

microbiology↗

bioRxiv2024

Temporal epigenome modulation enables efficient bacteriophage engineering and functional analysis of phage DNA modifications

Lytic bacteriophages hold substantial promise in medical and biotechnological applications. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.

microbiology↗

bioRxiv2024

Identification, structure and function of the methyltransferase involved in the biosynthesis of the dithiolopyrrolone antibiotic xenorhabdin

Xenorhabdins (XRDs) are produced by Xenorhabdus species and are members of the dithiopyrrolone (DTP) class of natural products that have potent antibacterial, antifungal and anticancer activity. The amide moiety of their DTP core can be methylated or not to fine-tune the bioactivity properties. However, the enzyme responsible for the amide N-methylation remained elusive. Here, we identified and characterized the amide methyltransferase XrdM that is encoded nearly 600 kb away from the XRD gene cluster using proteomic analysis, methyltransferase candidate screening, gene deletion, and allied approaches. In addition, crystallographic analysis and site-directed mutagenesis proved that XrdM is completely distinct from the recently reported DTP methyltransferase DtpM, and that both have been tailored in a species-specific manner for DTP biosynthesis in Gram-negative/positive organisms. Our study expands the limited knowledge of post-NRPS amide methylation in DTP biosynthesis and reveals the evolution of two structurally completely different enzymes for the same reaction in different organisms.

microbiology↗

bioRxiv2023

A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material

BackgroundKomagataella phaffii (Pichia pastoris) is a methylotropic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers. ResultsHere, we describe a linear "nano"chromosome (of 15-25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system. ConclusionsWe have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

bioengineering↗

bioRxiv2023

DNA replication is not a limiting factor for the rapid growth of Vibrio natriegens

DNA replication prior to cell division is essential for the proliferation of all cells. Bacterial chromosomes are replicated bidirectionally from a single origin of replication, with replication proceeding at about 1000 bp per second. For the best-studied model organism, Escherichia coli, this translates into a replication time of about 40 min for its 4.6 Mb chromosome. Nevertheless, E. coli can propagate by overlapping replication cycles with a maximum short doubling time of 20 min. The fastest growing bacterium known today, Vibrio natriegens, is able to replicate with a generation time of less than 10 min. It has a bipartite genome with chromosome sizes of 3.2 and 1.9 Mb. Is simultaneous replication from two origins a prerequisite for its rapid growth? We fused the two chromosomes of V. natriegens to create a strain carrying a 5.2 Mb chromosome with a single origin of replication. Compared to the wild-type, this strain showed little deviation in growth rate. This suggests that the split genome is not a prerequisite for rapid growth, and that DNA replication is not an important growth rate-limiting factor.

microbiology↗

bioRxiv2023

Large-scale genomic rearrangements boost SCRaMbLE in Saccharomyces cerevisiae

Genomic rearrangements contribute to gene copy number alterations, disruption of protein-coding sequences and/or perturbation of cis-regulatory networks. SCRaMbLE, a Cre/loxP-based system implanted in synthetic yeast chromosomes, can effectively introduce genomic rearrangements, and is thus a potential tool to study genomic rearrangements. However, the potential of SCRaMbLE to study genomic rearrangements is currently hindered, because a strain containing all 16 synthetic chromosomes is not yet available. Here, we constructed a yeast strain, SparLox83, containing 83 loxPsym sites distributed across all 16 chromosomes, with at least two sites per chromosome. Inducing Cre recombinase expression in SparLox83 produced versatile genome-wide genomic rearrangements, including inter-chromosomal events. Moreover, SCRaMbLE of the hetero-diploid strains derived from crossing SparLox83 with strains possessing synthetic chromosome III (synIII) from the Sc2.0 project led to increased diversity of genomic rearrangements and relatively faster evolution of traits compared to a strain with only synIII. Analysis of these evolved strains demonstrates that genomic rearrangements can perturb the transcriptome and 3D genome structure and can consequently impact phenotypes. In summary, a genome with sparsely distributed loxPsym sites can serve as a powerful tool to study the consequence of genomic rearrangements and help accelerate strain engineering in Saccharomyces cerevisiae.

synthetic biology↗

bioRxiv2023

Differential regulation of Retinoic Acid Metabolism in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic disease characterized by heterogeneous congenital abnormalities and increased risk for bone marrow failure and cancer. FA is caused by mutation of any one of 23 genes, the protein products of which function primarily in the maintenance of genome stability. An important role for the FA proteins in the repair of DNA interstrand crosslinks (ICLs) has been established in vitro. While the endogenous sources of ICLs relevant to the pathophysiology of FA have yet to be clearly determined, a role for the FA proteins in a two-tier system for the detoxification of reactive metabolic aldehydes has been established. To discover new metabolic pathways linked to FA, we performed RNA-seq analysis on non-transformed FA-D2 (FANCD2-/-) and FANCD2-complemented patient cells. Multiple genes associated with retinoic acid metabolism and signaling were differentially expressed in FA-D2 (FANCD2-/-) patient cells, including ALDH1A1 and RDH10, which encode for retinaldehyde and retinol dehydrogenases, respectively. Increased levels of the ALDH1A1 and RDH10 proteins was confirmed by immunoblotting. FA-D2 (FANCD2-/-) patient cells displayed increased aldehyde dehydrogenase activity compared to the FANCD2-complemented cells. Upon exposure to retinaldehyde, FA-D2 (FANCD2-/-) cells exhibited increased DNA double-strand breaks and checkpoint activation indicative of a defect in the repair of retinaldehyde-induced DNA damage. Our findings describe a novel link between retinoic acid metabolism and FA and identify retinaldehyde as an additional reactive metabolic aldehyde relevant to the pathophysiology of FA.

biochemistry↗

bioRxiv2022

L-SCRaMbLE creates large-scale genome rearrangements in synthetic Sc2.0 chromosomes

Optimization of the metabolic flux through heterologous pathways to improve bioproduction or utilization of alternative substrates requires both fine-tuning of non-native gene expression levels and improvement of the host genome. The SCRaMbLE system incorporated into synthetic Sc2.0 yeast strains enables a rapid approach to rearrange the genome of Saccharomyces cerevisiae in order to create optimized chassis. Here, we show that the light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or circular synthetic chromosome III. We present an efficient and straightforward workflow for the identification of complex rearranged synthetic chromosomes from SCRaMbLEd isolates without selection pressure. The screening method is based on novel genotyping primers, the loxPsym tags, which indicate not only deletions but also inversions and translocations. Long-read Nanopore sequencing is used to decode the selected genotypes and shows in conjunction with flow cytometry that large-scale karyotype alterations can be a consequence of SCRaMbLE.

synthetic biology↗

bioRxiv2022

Reconstruct a eukaryotic chromosome arm by de novo design and synthesis

The genome of an organism is inherited from its ancestor and keeps evolving over time, however, how much the current version could be altered remains unknown. Here, we use the left arm of chromosome XII (chrXIIL) as an example to probe the genome plasticity in Saccharomyces cerevisiae. A neochromosome was designed to harbor originally dispersed genes. The essentiality of sequences in chrXIIL was dissected by targeted DNA removal, chromosome truncation and random deletion. Notably, 12 genes were sufficient for survival, while 25 genes are required to retain robust fitness. Next, we demonstrated these genes could be reconstructed using synthetic regulatory sequences and recoded open-reading frames with "one-amino-acid-one-codon" strategy. Finally, we built a neochromsome, which could substitute for chrXIIL for cell viability, with these reconstructed genes. Our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional chromosomes with completely artificial sequences. HIGHLIGHTSO_LIA neochromosome was designed to facilitate the assembly of exogenous DNA for stable expression in yeast C_LIO_LIThe left arm of chrXII could be minimized to just 12 genes to maintain viability, but additional genes were required to retain robust fitness C_LIO_LIComprehensive recoding and transcriptional refactoring using artificial regulatory sequences produced a functional chromosome arm C_LIO_LIA completely reconstructed neochromosome could replace the chrXIIL to maintain comparable fitness C_LI

synthetic biology↗

bioRxiv2022

Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast

Here we report the design, construction and characterization of a tRNA neochromosome, a designer chromosome that functions as an additional, de novo counterpart to the native complement of Saccharomyces cerevisiae. Intending to address one of the central design principles of the Sc2.0 project, the [~]190 kb tRNA neochromosome houses all 275 relocated nuclear tRNA genes. To maximize stability, the design incorporated orthogonal genetic elements from non-S. cerevisiae yeast species. Furthermore, the presence of 283 rox recombination sites enable an orthogonal SCRaMbLE system capable of adjusting tRNA abundance. Following construction, we obtained evidence of a potent selective force once the neochromosome was introduced into yeast cells, manifesting as a spontaneous doubling in cell ploidy. Furthermore, tRNA sequencing, transcriptomics, proteomics, nucleosome mapping, replication profiling, FISH and Hi-C were undertaken to investigate questions of tRNA neochromosome behavior and function. Its construction demonstrates the remarkable tractability of the yeast model and opens up new opportunities to directly test hypotheses surrounding these essential non-coding RNAs. HighlightsO_LIDe novo design, construction and functional characterization of a neochromosome containing all 275 nuclear tRNA genes of Saccharomyces cerevisiae. C_LIO_LIIncreasing the copy number of the 275 highly expressed tRNA genes causes cellular burden, which the host cell likely buffers either by selecting for partial tRNA neochromosome deletions or by increasing its ploidy. C_LIO_LIThe tRNA neochromosome can be chemically extracted and transformed into new strain backgrounds, enabling its transplantation into multi-synthetic chromosome strains to finalize the Sc2.0 strain. C_LIO_LIComprehensive functional characterization does not pinpoint a singular cause for the cellular burden caused by the tRNA neochromosome, but does reveal novel insights into its tRNA and structural chromosome biology. C_LI

synthetic biology↗