Biology subjects

Waldburger, L.

Publications and source records attributed to Waldburger, L..

8 recordsLinked to original sources

bioRxiv2025

Binary vector origin predictably determines Agrobacterium-mediated transformation outcome across eukaryotic kingdom

Agrobacterium-mediated transformation (AMT) is the primary means of genetic engineering in plants and many fungi, but the factors that control transformation outcomes--efficiency, transgene insertion number, and transgene integrity--remain poorly characterized. Although transformation outcomes dictate an events potential utility in both industrial and academic contexts, AMT remains largely unoptimized for these metrics. Here, we systematically analyze the impact of the transgene-harboring binary vector on transformation outcomes across plant and fungal species. Through a comparison of different plasmid origin of replication (ORI) families and engineered copy number variants, our results reveal that the ORI family--not plasmid copy number--dictates T-DNA insertion number, backbone inclusion, and transformation efficiency, while plasmid copy number tuning alters efficiency without changing ORI family-specific signatures. Independent of plasmid copy number across kingdoms, the most widely used pVS1 ORI-based vectors (e.g. pCambia) result in significantly more insertions per transformant and high levels of transgene silencing compared to the less-utilized pSa ORI family, which enriches for more uniform single insertion events. Furthermore, we demonstrate that ORI-dependent transformation outcomes in yeast predictably reflect those in Arabidopsis. Together, these results lay the foundation for future binary vector design aimed at achieving more predictable, controllable, and optimized transformation outcomes across diverse eukaryotic hosts.

plant biology↗

bioRxiv2025

Diversity-driven biochemical survey reveals dimeric structural origin of rubisco

Rubisco is the entry point of nearly all organic carbon into the biosphere and is present in all domains of life. Despite its global importance, biochemical studies of this enzyme superfamily have been limited to a relatively narrow set of subclades. Recent advances in metagenomics have dramatically reshaped our understanding of both microbial and rubisco diversity; however, biochemical characterization of these sequences has not kept pace with the exponential growth in sequence data. To better survey the functional and structural diversity of rubisco, we systematically sampled and synthesized a library of diverse rubisco sequences with an emphasis on clades that have previously not been characterized. Our updated phylogenetic analysis reveals that many deep-branching rubiscos assemble as dimers, supporting a dimeric origin for the superfamily -- in contrast to the ecologically dominant hexadecameric form I. Additionally, we discover and structurally characterize the largest rubisco described to date, originating from a cryptic, early-branching subclade with novel structural folds that have previously not been observed in the rubisco superfamily. By integrating biochemical data with an updated phylogenetic framework, we propose a revised nomenclature for the rubisco protein family that reflects current insights and will better accommodate future discoveries.

biochemistry↗

bioRxiv2025

Active learning enables discovery of transcriptional activators across fungal evolutionary space

Biological discovery and design are increasingly being guided by predictive models in place of costly experimentation. However, existing datasets are often biased by overrepresentation from model organisms, leading to failures in evolutionary studies of non-model species. We present a hybrid framework that leverages high-throughput molecular assays and active learning to quantify biological properties across evolutionary space. We focus on transcriptional activators, which contain activation domains (ADs) that promote gene expression. ADs are intrinsically disordered and poorly conserved, which limits their study using comparative genomics. Here, we developed ADhunter, a high-capacity regression model that outperforms state-of-theart algorithms in identifying and quantifying the strength of transcriptional activators. Model uncertainty was used to guide evolutionary sampling across 7.8 million proteins from 2,400 fungal genomes. We functionally characterized 9,836 ADs from 1,071 fungal genomes, providing a 15.5-fold expansion in genome representation compared to existing datasets. Comprehensive sampling from non-model genomes improved model generalizability and provides the first functional annotation for 3,416 proteins from 670 non-model fungi. Model interpretability analysis aligns with the biophysical model of AD function and reveals novel, underrepresented protein codes, highlighting the importance of sampling from non-model organisms to build evolutionarily robust models for predicting biological properties.

genomics↗

bioRxiv2025

Multiplexed profiling of transcriptional regulators in plant cells

Transcriptional regulators play key roles in plant growth, development, and environmental responses; however, understanding how their regulatory activity is encoded at the protein level has been hindered by a lack of multiplexed large-scale methods to characterize protein libraries in planta. Here, we present ENTRAP-seq (Enrichment of Nuclear Trans-elements Reporter Assay in Plants), a high-throughput method that introduces protein-coding libraries into plant cells to drive a nuclear magnetic sorting-based reporter, enabling multiplexed measurement of regulatory activity from thousands of protein variants. Using ENTRAP-seq and machine learning, we screened 1,495 plant viruses and identified hundreds of novel putative transcriptional regulatory domains found in structural proteins and enzymes not associated with gene regulation. In addition, we combined ENTRAP-seq with machine-guided design to engineer the activity of a plant transcription factor in a semi-rational fashion. Our findings demonstrate how scalable protein function assays deployed in planta will enable the characterization of natural and synthetic coding diversity in plants.

plant biology↗

bioRxiv2025

Leveraging a synthetic biology approach to enhance BCG-mediated expansion of Vγ9Vδ2 T cells

There is an urgent need to develop a more efficacious anti-tuberculosis vaccine as the current live-attenuated vaccine strain BCG fails to prevent pulmonary infection in adults. Our long-term goal is to test whether increasing the immunogenicity of BCG will improve vaccine effectiveness while maintaining its proven safety profile. In this study, we leverage a synthetic biology approach to engineer BCG to produce more (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a phosphoantigen produced as an intermediate of bacterial--but not host--isoprenoid biosynthesis via the methylerythritol phosphate (MEP) pathway. Importantly, HMBPP strongly activates and expands V{gamma}9V{delta}2 T cells, which are unique to higher-order primates and protect against Mycobacterium tuberculosis infection. Prior work to engineer BCG to produce specific ligands and antigens has been attempted to some success; however, our strategy exploits a self-nonself recognition mechanism in the host via HMBPP sensing, which has not been attempted before in this way. To inform the design of our recombinant strains, we performed synteny analyses of >63 mycobacterial species, which revealed that isoprenoid biosynthetic genes are not found in gene clusters or operons across all the 356 surveyed genomes. This analysis also revealed pair biases of isoprenoid biosynthesis genes frequently found in close proximity. In our engineering attempts, we found that simply overexpressing the rate-limiting gene in the pathway was toxic to the bacterium. Thus, we generated synthetic loci with the goal of specifically overproducing HMBPP, and tested the ability of these engineered strains to induce human V{gamma}9V{delta}2 expansion in an in vitro stimulation assay. We found that BCG expressing a rationally-designed, synthetic MEP locus did not enhance V{gamma}9V{delta}2 T cell expansion over the wild-type vaccine strain, suggesting that ectopic expression of multiple MEP genes may result in feedback inhibition of the pathway. However we found that overexpression of the HMBPP synthase GcpE alone potently induced V{gamma}9V{delta}2 T cell expansion and did not result in downregulation of other pathway genes, presenting a successful strategy to accumulate HMBPP and overcome feedback inhibition in this pathway. While much remains to be done to ultimately develop a more efficacious vaccine, our data present a promising system to improve upon the BCG platform. To our knowledge, this is the first work to attempt reengineering of the MEP pathway in BCG to improve vaccine efficacy.

bioengineering↗

bioRxiv2024

AAV Capsid Screening for Translational Pig Research Using a Mouse Xenograft Liver Model

In gene therapy, delivery vectors are a key component for successful gene delivery and safety, based on which adeno-associated viruses (AAVs) gained popularity in particular for the liver, but also for other organs. Traditionally, rodents have been used as animal models to develop and optimize treatments, but species and organ specific tropism of AAV desire large animal models more closely related to humans for preclinical in-depth studies. Relevant AAV variants with the potential for clinical translation in liver gene therapy were previously evolved in vivo in a xenogeneic mouse model transplanted with human hepatocytes. Here, we selected and evaluated efficient AAV capsids using chimeric mice with a >90% xenografted pig hepatocytes. The pig is a valuable preclinical model for therapy studies due to its anatomic and immunological similarities to humans. Using a DNA-barcoded recombinant AAV library containing 47 different capsids and subsequent Illumina sequencing of barcodes in the AAV vector genome DNA and transcripts in the porcine hepatocytes, we found the AAVLK03 and AAVrh20 capsid to be the most efficient delivery vectors regarding transgene expression in porcine hepatocytes. In attempting to validate these findings with primary porcine hepatocytes, we observed capsid-specific differences in cell entry and transgene expression efficiency where the AAV2, AAVAnc80, and AAVDJ capsids showed superior efficiency to AAVLK03 and AAVrh20. This work highlights intricacies of in vitro testing with primary hepatocytes and the requirements for suitable pre-clinical animal models but suggests the chimeric mouse to be a valuable model to predict AAV capsids to transduce porcine hepatocytes efficiently.

molecular biology↗

bioRxiv2023

The pGinger family of expression plasmids

The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of RFP, with a broad host range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry.

synthetic biology↗

bioRxiv2022

Borg tandem repeats undergo rapid evolution and are under strong selection to create new intrinsically disordered regions in proteins

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of tandem repeats in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently and are virtually absent in host Methanoperedens genomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversify Methanoperedens and all TRs are crucial for specific Borg-host associations and possibly co-speciation.

microbiology↗