Schedule
| Mar 13th | Mar 14th | Mar 15th |
| 1:00 PM – |
1:00 PM Henry Lee |
1:00 PM Daniel Stukenberg |
| 1:15 PM Tianyu Li |
1:30 PM Jorg Soppa |
1:30 PM Anna Faber |
| 1:45 PM Hyun Gyu Lim |
1:45 PM Richard Biener |
1:45 PM David Specht |
| 2:15 PM Yungyu Lee |
2:00 PM Break Time |
2:15 PM B Thuronyi |
| 2:30 PM Xiang Gao |
2:10 PM Terry Hwa |
2:45 PM Break Time |
| – | – | 2:55 PM Tanya Tschirhart |
| – | – | 3:25 PM Jongoh Shin |
Talk times are in UTC and should be regarded as tentative.
Abstracts
March 13th : Vnat Applications
Tianyu Li
Engineering Vibrio natriegens for Degrading and Assimilating Poly (Ethylene Terephthalate)
North Carolina State University
Poly(ethylene terephthalate) (PET), a widely used and recyclable plastic, poses environmental challenges due to its resistance to natural degradation. Despite various recycling strategies, the downcycled products often end up in landfills, leading to the formation of microplastics. This accumulation of landfilled PET waste poses a significant threat to marine life, ecosystems, and potentially human health. To address this issue, our project leveraged synthetic biology to develop a whole-cell biocatalyst capable of depolymerizing PET in seawater environments and growing on its breakdown products, using the fast-growing, nonpathogenic, moderate halophile Vibrio natriegens. By leveraging a two-enzyme system – comprising a chimera of IsPETase and IsMHETase from Ideonella sakaiensis – displayed on Vibrio natriegens, we constructed whole-cell catalysts that depolymerize PET and convert it into its monomers in salt-containing media and at a temperature of 30 °C. Finally, Vibrio natriegens has been further engineered to utilize terephthalic acid (TPA), one of the degradation products of PET, as a sole carbon source through metabolic engineering. Vibrio natriegens isolates that exhibit stable growth in minimal medium supplemented with TPA as the sole carbon source have been identified and characterized. Taken together, this work introduces bioremediation of PET plastics in brackish water, thereby improving process sustainability.
Hyun Gyu Lim
Engineering Vibrio sp. dhg for Accelerated Biorefineries
Inha University
The development of efficient microbial processes for producing biochemicals from biomass has become a critical issue due to concerns about climate change and the depletion of fossil fuels. Host microorganisms’ inherent metabolic activities significantly impact process efficiency, making it vital to use microorganisms that can quickly convert biomass-derived ugars. In this study, we report Vibrio sp. dhg as an efficient microbial platform that can consume major sugars from marine and lignocellulosic biomass faster than any previously reported microorganisms. We developed a genetic toolbox through systematic genomic characterization, allowing for its engineering. Furthermore, we enhanced sugar catabolism using a combination of rational and evolutionary engineering. We successfully demonstrated the strain’s ability to rapidly produce ethanol, 2,3-butanediol, lycopene, and lactic acid from biomass sugar mixtures with high productivities and yields. Our findings suggest that this strain can be broadly used to expedite the production of diverse biochemicals from different types of biomass.
Yungyu Lee
Natural Transformation of Vibrio natriegens with Large Genetic Cluster Enables Alginate Assimilation in Isopentenol Production
Seoul National University
Alginate is a major component of brown macroalgae, and its efficient utilization is critical for the development of sustainable technologies where abundant marine biomass could be converted into valuable products. Vibrio natriegens is a fast-growing marine-based bacterium that has gained huge attention in the biotechnological scene due to its potential as an alternative industrial chassis. However, V. natriegens cannot naturally metabolize alginate, limiting its usage in marine biomass conversion. In this study, we engineered V. natriegens to utilize crude marine biomass, kelp, as a carbon source. 33kb of the genetic cluster from Vibrio sp. dhg was integrated into V. natriegens through natural transformation, a unique DNA uptake mechanism possessed by Vibrio species, enabling alginate metabolism. We further demonstrated the potential of this engineered V. natriegens strain for the production of isopentenol, a valuable chemical used in the production of fuels, plastics, and other products. Our final strain produced 1.8mg L-1 of isopentenol from direct fermentation of kelp powder. This study not only presents the very first case in which V. natriegens is naturally transformed with large DNA fragments ranging up to 33kb but also highlights the potential of this engineered strain for the conversion of marine biomass into valuable products.
Xiang Gao
Solar-driven Waste-to-chemical Conversion by Wastewater-derived Semiconductor Biohybrids
Shenzhen Institutes of Advanced Technology, Chinese Academic of Sicence
Semiconductor biohybrids, integrating the merits of living cells and semiconductor materials, have the potential to shift the current energy-intensive chemical production system to a more sustainable one by offering efficient solar-to-chemical conversion. However, cost-competitive and environmentally friendly scaling-up approaches are still in urgent need. To tackle this challenge, we propose a strategy that co-utilize pollutants in wastewater to produce semiconductor biohybrids in-situ for scalable solar-to-chemical conversion. Specifically, we introduced an aerobic sulfate reduction pathway into Vibrio natriegens to enable the direct utilization of heavy metal ions (i.e., Cd²⁺), sulfate, and organics in wastewater to biosynthesize functional semiconductor nanoparticles in living V. natriegens, to assemble semiconductor biohybrids. Meanwhile, a designated biosynthetic pathway was introduced into the biohybrids to enable the production of 2,3-butanediol, a valuable bulk chemical with wide applications, from organics in wastewater. Using the obtained biohybrids, the production of 2,3-butanediol reached 13.09 g L-1 in a 5 L illuminated fermenter using wastewater as the feedstock, revealing its scalability. Life cycle assessment showed this specific biohybrid route had substantial sustainability gain, compared to conventional 2,3-butanediol production routes. This work can bring solar-driven biomanufacturing and waste-to-wealth conversion one step forward, and pave the way to cleaner production and circular economy.
March 14th : Vnat Basic Science
Henry Lee
(Community Needs)
Cultivarium
TBA
Jorg Soppa
Ploidy in Vibrio natriegens: Very dynamic and Rapidly Changing Copy Numbers of Both Chromosomes
Goethe-University, Frankfurt, Germany
TBA
Richard Biener
Low-chloride Chemically Defined Media for Vibrio natriegens
University of Applied Sciences Esslingen
Due to its fast generation time, Vibrio natriegens has high potential as a production strain for biotechnological production processes or other applications in biotechnology. Culture media for V. natriegens typically contain high concentrations of sodium chloride. The corresponding high chloride concentrations can lead to corrosion processes on metal surfaces in bioreactors. Here we report the development of a defined medium for V. natriegens with low chloride concentration. Sodium chloride was completely replaced by the sodium salts disodium hydrogen phosphate, disodium sulfate and sodium citrate, while keeping the total concentration of sodium ions constant. The use of citrate prevents precipitations, especially of ammonium magnesium phosphate. With this defined medium, high cell density fed-batch cultivations in lab-scale bioreactors (1 L to 20 L) yielded biomass concentrations of more than 80 gL-1. The chemically defined low-chloride media developed can realize high biomass yields while preventing corrosion of the bioreactor steel. This will allow the strain to reach its full potential for industrial applications in the future.
Terry Hwa
Physiological Characteristics of V. natriegens
UC San Diego
In this talk, i will compare to the physiological characteristics of Vnat with other model bacteria such as E. coli and B. sutbilis, with focus on what allows Vnat to grow so fast.
March 15th : Vnat Engineering Tools
Daniel Stukenberg
Tool Development for V. natriegens
Vibrio natriegens is the fastest-growing organism known to date with a doubling time of under 10 minutes. This rapid growth can potentially be exploited shorten bacterial incubation times drastically, thereby accelerating scientific progress in general. To turn a fast-growing bacterium isolated from salt marshes into a genetically tractable SynBio chassis, we developed the necessary tools. First, we established NT-CRISPR, which combines genome engineering by natural transformation with CRISPR-Cas9-based counterselection. As a result, NT-CRISPR allows us to efficiently perform deletions, integrations, and point mutations in a scarless and markerless fashion. Furthermore, we demonstrated the simultaneous deletion of three distinct chromosomal regions. Second, we established graded-CRISPRi as a novel tool to regulate expression of chromosome encoded genes in V. natriegens. Through inducible expression of both cas9 and gRNA, we created a tightly controlled CRISPRi system while allowing strong repression of the target gene after addition of inducers. Additionally, we used gRNA libraries with mismatches to the target sequence to achieve graded repression levels. This allows the investigation of protein-abundance-dependent phenotypes for both essential and non-essential genes.
Anna Faber
The Vnat2.0 collection: An Enhanced and Expanded Golden Gate Toolkit for Vibrio natriegens
The University of Western Australia
With its remarkable doubling time of less than 10 minutes, V. natriegens presents an opportunity to significantly accelerate the cycle of design-build-test-learn in synthetic biology. However, the full potential of V. natriegens as a synthetic biology platform is highly dependent on the availability of specialized genetic tools tailored to its unique biology. Therefore, the development of synthetic biology toolboxes, like the Marburg Collection (Stukenberg et al., 2021), is crucial for unlocking the capabilities of V. natriegens. The Marburg Collection is a Golden Gate cloning toolbox, comprising standardized genetic parts designed for modularity and ease of assembly. Our recent collaborative work has focused on creating a significant expansion of this collection for V. natriegens. We curated selected overhangs of the original Marburg Collection, tested an improved design for dropout parts, and built a selection software for easier part management. Furthermore, we characterized additional inducible promoters, novel operon connectors, and homology flanks for NT-CRISPR. Hence, we present a toolkit upgrade for genetic engineering in V. natriegens which enhances the efficiency, number, and quality of available parts. By establishing this collection expansion, we contribute to the ongoing advancement of genetic tools and their cross compatibility for V. natriegens to increase the genetic accessibility of this promising chassis.
David Specht
Engineering Enhanced Natural Competence in V. natriegens using CRISPR-associated Transposons
Cornell University
CRISPR-Associated Transposon (CAST) systems are a powerful tool for the genomic insertion of arbitrary large cargos into diverse microbes, including V. natriegens. Using a CAST system, we are building a full genome knockout collection in V. natriegens using Knockout Sudoku, a method used to spatially resolve tens of thousands of mutants produced via transposon mutagenesis. Use of a targeted transposon system, rather than random insertions (as is done in the original Knockout Sudoku), enables rapid production of select sets and combinations of mutants, as well as the generation of an upregulation collection, resources which we believe could be invaluable to the V. natriegens community. We are applying this tool particularly to the enhancement of natural competence to plasmids, the native ability of V. natriegens to take up intact DNA from its environment, which has implications for the accessibility of V. natriegens research and the democratization of synthetic biology more broadly. Enhancement of natural competence to the point of unconditional DNA uptake could enable radical genetic engineering in V. natriegens and transient DNA expression for the creation of a true biological computer.
B Thuronyi
Advancing Vibrio natriegens as a model organism for studying plasmid natural transformation
Williams College
Horizontal gene transfer among bacteria shapes their evolution, ecology, and human health impacts, and can also be leveraged for synthetic biology. Plasmids are important in this process because they replicate autonomously and sometimes with broad host range. While plasmid transfer by conjugation is well studied, natural transformation of plasmids has been comparatively neglected. Uniquely, this mechanism operates on naked DNA and is fully directed by the recipient cell. Its importance in nature and its biotechnological potential are underexplored. Vibrio natriegens is highly naturally competent, and modification of its genome through natural transformation is already a vital tactic for engineering. We are working to position V. natriegens as a tractable, safe, and convenient model system for quantitative study of natural plasmid transformation. The repertoire of plasmids available for this Gram-negative host is large and its growth rate and competence properties make it well suited to comparative transformation efficiency studies. We are establishing reproducible and scalable methods for production of plasmid concatemers that support high transformation efficiency and validating accurate and low-variance plating techniques for use with V. natriegens. Using these approaches, we are investigating plasmid sequence determinants of transformation efficiency to illuminate the molecular mechanisms of the process.
Tanya Tschirhart
Genome Engineering and Reduction in Vibrio natriegens
U. S. Naval Research Laboratory
The fast-growing marine bacterium Vibrio natriegens has recently garnered attention as a host for molecular biology and biotechnology applications despite the fact that comparatively little is known about the basic biology of this non-model organism. Herein, we will describe our efforts to develop genome engineering and reduction tools to better understand and utilize this promising chassis. In our approach, we aim to utilize -omics data and systems-level responses of V. natriegens in various conditions to guide genome engineering. We have developed tools for insertions of whole transcriptional units into the genome, which will be hugely helpful for scale up of biomanufacturing. We have also utilized a genome reduction tool to create large chromosomal deletions in V. natriegens. The rich data from the analysis of these engineered strains will aid ongoing and future bioproduction efforts which take advantage of the outstanding characteristics of this organism.
Jongoh Shin
Systems Biology of Competency in Vibrio natriegens
UC San Diego
The natural competence of Vibrio natriegens is orchestrated by the TfoX and QstR transcription factors, crucial for the uptake and transport of external DNA. Despite this understanding, the broader genetic and transcriptional regulatory mechanisms underpinning this competency are still largely unexplored. In this study, we employed a machine-learning method to dissect the transcriptome of Vibrio natriegens, identifying 45 independent modulons of gene regulation, termed iModulons. Our analysis reveals that natural competence is linked to the suppression of two housekeeping iModulons (related to iron metabolism and translation) and the activation of six iModulons. These include the known TfoX and QstR, a newly identified iModulon of uncertain function, and three housekeeping iModulons associated with motility, polycations, and reactive oxygen species (ROS) responses. Further, phenotypic analysis of 83 gene deletion strains indicated that disrupting iModulon functionality significantly diminishes or abolishes natural competence. This iModulon discovery sheds light on the transcriptomic landscape of natural competence and its interplay with basic cellular functions, offering a genetic framework for the systems biology of competency in Vibrio natriegens.