Biology subjects

Schubert, M. G.

Publications and source records attributed to Schubert, M. G..

8 recordsLinked to original sources

bioRxiv2026

Bioplastic Production from simulated 100% in situ Mars resources

A sustained human presence on Mars requires local production of bulk materials, particularly polymers. Prior approaches to space biomanufacturing rely on human waste streams, Earth-sourced consumables, or complex chemical infrastructure, limiting their ability to scale. Here we demonstrate production of polyhydroxyalkanoate (PHA) bioplastic from simulated 100% martian resources: regolith-derived soluble nutrients, acetate electrochemically fixed from martian atmosphere, and water. We screened 16 candidate organisms for growth in a chemically defined Mars medium and identified Cupriavidus necator H16 and Pseudomonas putida KT2440 as promising chassis organisms. Adaptive laboratory evolution totaling more than 1012 cumulative cell divisions improved both species' tolerance to high concentrations of acetate and leached regolith. The top C. necator evolved isolate, referred to as sPL.001, produced more than three-fold higher PHA titer under simulated Mars conditions compared to its parent strain. These results establish a path to polymer production on Mars where consumable mass is derived entirely from local resources, decoupling bulk material production from Earth supply chains.

bioengineering↗

bioRxiv2026

Defined Mars Media (DMM), a chemically defined simulant of the soluble macro- and micro- nutrients in Mars regolith for use in biological research

Mars harsh yet workable surface conditions, such as manageable temperatures, availability of solar energy, and in situ resources like water ice, carbon dioxide, and mineral-rich regolith, make it a compelling target for supporting life beyond Earth. However, existing experiments testing chemical habitability in Mars conditions generally rely on leachates of physical regolith simulants, which vary in composition across simulant types, leaching conditions, and production batches. We introduce a defined Mars media (DMM) that accurately simulates the biologically relevant nutrients (nitrogen, phosphorus, and sulfur) and stressors (perchlorates, heavy metals) in Martian regolith when it is leached in water at neutral pH. We formulated DMM by combining direct rover and lander measurements from Mars with laboratory measurements of regolith simulant leachates. We validate DMM from a 1x to 20x concentrate, equivalent to 40 g/L to 800 g/L of leached regolith. Using DMM with acetate as a Mars atmosphere-derived carbon source, we grew eight bacteria, demonstrating that organisms can source all essential nutrients from Martian resources. We also demonstrate that microbial growth in DMM is robust to uncertainties in Martian regolith composition: sensitivity experiments can identify limiting trace element nutrients and toxins in DMM, and show that bacterial growth is maintained across at least an order of magnitude variation in their concentrations. This is the first defined Mars regolith media recipe containing both macro- and micro-nutrients, and designed specifically for biological experimentation. By shifting from variable leachate-based approaches to a defined aqueous analog, we enable controlled hypothesis testing of microbial survival, growth, and function in a Martian chemical environment. DMM will enable further research on astrobiology, biological in situ resource utilization, large-scale soil remediation, and terraforming. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/719001v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@37408forg.highwire.dtl.DTLVardef@16fabfforg.highwire.dtl.DTLVardef@84fa43org.highwire.dtl.DTLVardef@4a7c5e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

BioBloom, a method for barcoded saturation mutagenesis of an entire bacterial genome

Saturation mutagenesis is a powerful tool for understanding and engineering the function of biological systems. and has been applied successfully to characterize the mutational landscape of individual proteins and genetic loci. However. it has not been applied at the whole-genome scale due to the challenges of both creating and quantifying a saturating set of mutations. Here we introduce BioBloom. a retron-based method for barcoded saturation mutagenesis at the scale of a whole bacterial genome. We constructed a barcoded BioBloom library with >99% projected sampling of saturating single - nucleotide polymorphism (SNP) mutations of the E. coli genome, and applied it to identify beneficial mutations under salt and antibiotic selection. Relative to other techniques like CRISPR-enabled mutagenesis or Adaptive Laboratory Evolution, BioBloom excels at identifying diverse causal SNPs quickly and at smaller working volumes. A barcoded, saturating mutation library is also a shared resource, and we are releasing the updated BioBloom-Ec.-2.0 library to the scientific community for broader adoption and application. Together. BioBloom makes barcoded saturation mutagenesis accessible at whole-genome scale, creating new opportunities for large-scale data collection and bacterial engineering.

microbiology↗

bioRxiv2024

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR/Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55 kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the WT. This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome to phenome relationships. ImportanceGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome editing tool and in this study we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

synthetic biology↗

bioRxiv2023

Cyanobacteria newly isolated from marine volcanic seeps display rapid sinking and robust, high density growth

Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling as well as promising bioproduction chassis. Here, we isolate two novel cyanobacteria, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO2. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically-relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (>31g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting improved de-watering of UTEX 3222 biomass. This settling behavior can be explained in part by larger cell volume. These traits may make UTEX 3222 a compelling choice for photosynthetic bioproduction from CO2. Overall, we find that bio-prospecting in environments with naturally elevated CO2 may uncover novel CO2-metabolizing organisms with unique characteristics.

microbiology↗

bioRxiv2021

Reconstructing transcriptional histories by CRISPR acquisition of retron-based genetic barcodes

Biological processes depend on the differential expression of genes over time, but methods to make physical recordings of these processes are limited. Here we report a molecular system for making time-ordered recordings of transcriptional events into living genomes. We do this via engineered RNA barcodes, based on prokaryotic retrons1, which are reverse-transcribed into DNA and integrated into the genome using the CRISPR-Cas system2. The unidirectional integration of barcodes by CRISPR integrases enables reconstruction of transcriptional event timing based on a physical record via simple, logical rules rather than relying on pre-trained classifiers or post-hoc inferential methods. For disambiguation in the field, we will refer to this system as a Retro-Cascorder.

synthetic biology↗

bioRxiv2020

Synthetic auxotrophy remains stable after continuous evolution and in co-culture with mammalian cells

Understanding the evolutionary stability and possible context-dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While batch cultures of synthetic auxotrophic Escherichia coli previously exhibited undetectable escape throughout 14 days of monitoring, the long-term effectiveness of synthetic auxotrophy is unknown. Here, we report automated continuous evolution of a synthetic auxotroph using custom chemostats that supply a decreasing concentration of essential biphenylalanine (BipA). After 100 days of evolution in three separate trials, populations exhibit no observable escape and are capable of normal growth rates at 10-fold lower BipA concentration than the ancestral synthetic auxotroph. Allelic reconstruction of three proteins implicated in small molecule transport reveals their contribution to increased fitness at low BipA concentrations. Mutations do not appear in orthogonal translation machinery nor in synthetic auxotrophic markers. Based on its evolutionary stability, we introduce the progenitor synthetic auxotroph directly to mammalian cell culture. We observe containment of bacteria without detrimental effects on HEK293T cells. Overall, our findings reveal that synthetic auxotrophy is effective on timescales and in contexts that enable diverse applications. One Sentence SummaryTo ascertain whether life inevitably finds a way, we continuously evolve an Escherichia coli strain that was not able to escape from engineered biocontainment before, and we find that it does not escape even after 100 days of evolution, nor does it escape when added to mammalian cell culture.

synthetic biology↗

bioRxiv2020

High throughput functional variant screens via in-vivo production of single-stranded DNA

Tremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

synthetic biology↗