More Like this

1. Combinatorial Metabolic Engineering of Alkane Biosynthesis in the Osmotolerant Yeast Debaryomyces hansenii CBS 767

   https://doi.org/10.1002/bit.70350  

   Li, Zekun; Sharma, Nilesh Kumar; Weintraub, Sarah; Young, Eric (August 2026, Biotechnology and Bioengineering)

   ABSTRACT Debaryomyces hanseniiis a yeast with diverse potential applications like producing oleochemicals from biomass. Yet, combinatorial metabolic engineering inD. hanseniiis limited because, like many nonconventional organisms, key genomic data and genetic elements are missing. Here, we report phenotypic characterization, genomic integration loci, and modular genetic parts that enable combinatorial metabolic engineering of alkane biosynthesis inD. hansenii. Phenotypic characterization revealed thatD. hanseniiproduces lipids when grown on components of lignocellulosic and algal biomass in standard and saline media. Notably,D. hanseniiproduced 57.25% more lipids thanYarrowia lipolyticaPo1f when grown on glucose. Next, genomic integration loci were identified and gene expression elements (using known homology toS. cerevisiae) were derived from theD. hanseniiCBS767 genome. We derived 23 promoters and 24 terminators, recoded known selection markers and reporters, designed genomic integration plasmids, and made all compatible with a modular cloning standard. Flow cytometry measurements show that the promoters span an expression range of three orders of magnitude and the terminators span one order of magnitude. To demonstrate combinatorial metabolic engineering, the parts collection was used to assemble 18 alkane biosynthesis pathways. With no other genetic modifications, the best strain produced 38.31 mg/L and 1.47 mg/L of heptadecane from glucose and arabinose, respectively. This work establishesD. hanseniias a cell factory for synthesizing oleochemicals from lignocellulose derived sugars while providing a blueprint for leveraging phenotyping, genomics, and modular parts collections with automation platforms to develop cell factories from nonconventional organisms. Furthermore, the genetic parts collection now enables functional genetics for various applications inDebaryomycesyeasts. 

   more » « less
2. Identifying widespread and recurrent variants of genetic parts to improve annotation of engineered DNA sequences

   https://doi.org/10.1371/journal.pone.0304164  

   McGuffie, Matthew J; Barrick, Jeffrey E (May 2024, PLOS ONE)

   Mienda, Bashir Sajo (Ed.)

   Engineered plasmids have been workhorses of recombinant DNA technology for nearly half a century. Plasmids are used to clone DNA sequences encoding new genetic parts and to reprogram cells by combining these parts in new ways. Historically, many genetic parts on plasmids were copied and reused without routinely checking their DNA sequences. With the widespread use of high-throughput DNA sequencing technologies, we now know that plasmids often contain variants of common genetic parts that differ slightly from their canonical sequences. Because the exact provenance of a genetic part on a particular plasmid is usually unknown, it is difficult to determine whether these differences arose due to mutations during plasmid construction and propagation or due to intentional editing by researchers. In either case, it is important to understand how the sequence changes alter the properties of the genetic part. We analyzed the sequences of over 50,000 engineered plasmids using depositor metadata and a metric inspired by the natural language processing field. We detected 217 uncatalogued genetic part variants that were especially widespread or were likely the result of convergent evolution or engineering. Several of these uncatalogued variants are known mutants of plasmid origins of replication or antibiotic resistance genes that are missing from current annotation databases. However, most are uncharacterized, and 3/5 of the plasmids we analyzed contained at least one of the uncatalogued variants. Our results include a list of genetic parts to prioritize for refining engineered plasmid annotation pipelines, highlight widespread variants of parts that warrant further investigation to see whether they have altered characteristics, and suggest cases where unintentional evolution of plasmid parts may be affecting the reliability and reproducibility of science. 

   more » « less
3. GoldenBraid2.0 E. coli : a comprehensive and characterized toolkit for enterics

   https://doi.org/10.1093/synbio/ysaf015  

   Cooke, Matthew B; Welch, Kobie T; Ramirez, Laura D; Wen, Alice X; Marciano, David C; Herman, Christophe (August 2025, Synthetic Biology)

   Billerbeck, Sonja (Ed.)

   Abstract Modular cloning systems streamline laboratory workflows by consolidating genetic ‘parts’ into reusable and modular collections, enabling researchers to fast-track strain construction. The GoldenBraid 2.0 modular cloning system utilizes the cutting property of type IIS restriction enzymes to create defined genetic ‘grammars’, which facilitate the reuse of standardized genetic parts and assembly of genetic parts in the right order. Here, we present a GoldenBraid 2.0 toolkit of genetic parts designed to accelerate cloning in the model bacterium Escherichia coli. This toolkit features 478 pre-made parts for gene expression and protein tagging as well as strains to expedite cloning and strain construction, enabling researchers to quickly generate functional plasmid-borne or chromosome-integrated expression constructs. In addition, we provide a complete laboratory manual with overviews of common reagent recipes, E. coli protocols, and community resources to promote toolkit utilization. By streamlining the assembly process, this resource will reduce the financial and temporal burdens of cloning and strain building in many laboratory settings. 

   more » « less
4. A modular plasmid toolkit applied in marine bacteria reveals functional insights during bacteria-stimulated metamorphosis

   https://doi.org/10.1128/mbio.01502-23  

   Alker, Amanda T.; Farrell, Morgan V.; Aspiras, Alpher E.; Dunbar, Tiffany L.; Fedoriouk, Andriy; Jones, Jeffrey E.; Mikhail, Sama R.; Salcedo, Gabriella Y.; Moore, Bradley S.; Shikuma, Nicholas J. (August 2023, mBio)

   Ruby, Edward G. (Ed.)

   ABSTRACT A conspicuous roadblock to studying marine bacteria for fundamental research and biotechnology is a lack of modular synthetic biology tools for their genetic manipulation. Here, we applied, and generated new parts for, a modular plasmid toolkit to study marine bacteria in the context of symbioses and host-microbe interactions. To demonstrate the utility of this plasmid system, we genetically manipulated the marine bacteriumPseudoalteromonas luteoviolacea, which stimulates the metamorphosis of the model tubeworm,Hydroides elegans. Using these tools, we quantified constitutive and native promoter expression, developed reporter strains that enable the imaging of host-bacteria interactions, and used CRISPR interference (CRISPRi) to knock down a secondary metabolite and a host-associated gene. We demonstrate the broader utility of this modular system for testing the genetic tractability of marine bacteria that are known to be associated with diverse host-microbe symbioses. These efforts resulted in the successful conjugation of 12 marine strains from the Alphaproteobacteria and Gammaproteobacteria classes. Altogether, the present study demonstrates how synthetic biology strategies enable the investigation of marine microbes and marine host-microbe symbioses with potential implications for environmental restoration and biotechnology. IMPORTANCEMarine Proteobacteria are attractive targets for genetic engineering due to their ability to produce a diversity of bioactive metabolites and their involvement in host-microbe symbioses. Modular cloning toolkits have become a standard for engineering model microbes, such asEscherichia coli, because they enable innumerable mix-and-match DNA assembly and engineering options. However, such modular tools have not yet been applied to most marine bacterial species. In this work, we adapt a modular plasmid toolkit for use in a set of 12 marine bacteria from the Gammaproteobacteria and Alphaproteobacteria classes. We demonstrate the utility of this genetic toolkit by engineering a marinePseudoalteromonasbacterium to study their association with its host animalHydroides elegans. This work provides a proof of concept that modular genetic tools can be applied to diverse marine bacteria to address basic science questions and for biotechnology innovations. 

   more » « less
5. MeMo: Meaningful, Modular Controllers via Noise Injection

   Tjandrasuwita, Megan; Xu, Jie; Solar-Lezama, Armando; Matusik, Wojciech (July 2024, ICML 2024)

   Robots are often built from standardized assemblies, (e.g. arms, legs, or fingers), but each robot must be trained from scratch to control all the actuators of all the parts together. In this paper we demonstrate a new approach that takes a single robot and its controller as input and produces a set of modular controllers for each of these assemblies such that when a new robot is built from the same parts, its control can be quickly learned by reusing the modular controllers. We achieve this with a framework called MeMo which learns (Me)aningful, (Mo)dular controllers. Specifically, we propose a novel modularity objective to learn an appropriate division of labor among the modules. We demonstrate that this objective can be optimized simultaneously with standard behavior cloning loss via noise injection. We benchmark our framework in locomotion and grasping environments on simple to complex robot morphology transfer. We also show that the modules help in task transfer. On both structure and task transfer, MeMo achieves improved training efficiency to graph neural network and Transformer baselines. 

   more » « less