Project description:Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology.
Project description:Producing the fuels and chemicals from renewable plant biomass has been thought as a feasible way for global sustainable development. However, the economical efficiency of biorefinery remains challenges. Here a cellulolytic thermophilic fungus, Myceliophthora thermophila, was constructed into a platform through metabolic engineering, which can efficiently convert lignocellulose to important bulk chemicals for polymers, four carbon 1, 4-diacids (malic and succinic acid), directly from lignocellulose without any extra enzymes addition or complicated pretreatment, with titer of over 200 g/L on cellulose and 110 g/L on plant biomass (corncob) during fed-batch fermentation. Our study represents a milestone of consolidated bioprocessing technology (CBP) and offers a new promising system for cost-effectively production of biomass-based chemicals and potentially fuels.
Project description:Genetic engineering of filamentous fungi has promise for accelerating the transition to a more sustainable food system and enhancing the nutritional value, sensory appeal, and scalability of microbial foods. However, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we developed a synthetic biology toolkit for Aspergillus oryzae, an edible fungus traditionally used in fermented foods and currently used in protein production and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for genome integration, neutral loci, and new promoters. We use these tools to enhance the elevate levels of the nutraceutical ergothioneine and intracellular heme in the edible biomass. The biomass overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of genetic approaches to enhance fungal meat alternatives and provide useful engineering tools for diverse applications in fungal food production and beyond.
Project description:In this project we aim to construct a tyrosine-producing E. coli strain through iterative steps of genome engineering. High PEP availability through knockout of the PTS was combined with the precise, in-place genomic integration of several engineering interventions, known to increase L-tyrosine production yields, to create a tyrosine-overproducing E. coli strain that can function as a platform for further engineering and optimization. Utilizing a design-build-test-learn (DBTL) cycle, an evolved pts-knockout E. coli strain was equipped with optimizations of the aroG, aroB and tyrA genes and cultivated under batch and fed-batch conditions. Subsequently, metabolomics, transcriptomics and proteomics samples from the fed-batch experiments were analyzed to inform the design of new genomic interventions.
Project description:Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high celluloseproducing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC.
Project description:To evaluate the biological impact of cellulose nanofibrils (CNFs) at the transcriptional level, we conducted whole-genome microarray analyses on human bronchial epithelial cells (BEAS-2B) exposed to CNFs with different physicochemical properties. The results were compared with those from exposures to microcrystalline cellulose (MCC).
Project description:To assess the biological impact of cellulose nanofibrils (CNFs) at the transcriptional level, we conducted whole-genome microarray analyses on rat alveolar macrophages (NR8383) exposed to CNFs with varying physicochemical properties. The findings were compared with those from exposure to microcrystalline cellulose (MCC) and lipopolysaccharide (LPS).
Project description:We compared the transcriptome of homozygous mutants for AtCesA4 and AtCesA6 (cellulose synthase genes) to their heterozygous counterparts that have a wild type phenotype. All plants were 4-weeks-old and grown under short day conditions.
Project description:Biomolecular condensates are membraneless compartments involved in a wide range of cellular processes. Despite their fundamental role in the spatiotemporal regulation of cellular functions, tools for precisely manipulating phase-separated condensates remain limited, and effective methods for discovering and functionalizing tunable phase separation modules from natural proteins are lacking. Here we present a rational engineering approach for androgen receptor (AR) and its clinically used drugs to create a chemical genetic platform, ARDrop, enabling condensates formation and dissolution. This platform is applied to a diverse set of proteins to achieve intended cellular functions, ensuring robust and long-lasting functionality through stable liquid-like properties. Our work develops a powerful toolkit for reversible manipulation of condensates that can be used for dissection of complicated cell signaling, laying the foundation for engineering designer condensates for synthetic biology applications.