Efficient yeast surface-display of novel complex synthetic cellulosomes

Background: The self-assembly of cellulosomes on the surface of yeast is a promising strategy for consolidated bioprocessing to convert cellulose into ethanol in one step. Results: In this study, we developed a novel synthetic cellulosome that anchors to the endogenous yeast cell wall protein a-agglutinin through disulfide bonds. A synthetic scaffoldin ScafAGA3 was constructed using the repeated N-terminus of Aga1p and displayed on the yeast cell surface. Secreted cellulases were then fused with Aga2p to assemble the cellulosome. The display efficiency of the synthetic scaffoldin and the assembly efficiency of each enzyme were much higher than those of the most frequently constructed cellulosome using scaffoldin ScafCipA3 from Clostridium thermocellum. A complex cellulosome with two scaffoldins was also constructed using interactions between the displayed anchoring scaffoldin ScafAGA3 and scaffoldin I ScafCipA3 through disulfide bonds, and the assembly of secreted cellulases to ScafCipA3. The newly designed cellulosomes enabled yeast to directly ferment cellulose into ethanol. Conclusions: This is the first report on the development of complex multiple-component assembly system through disulfide bonds. This strategy could facilitate the construction of yeast cell factories to express synergistic enzymes for use in biotechnology

Modular Synthesis of Functionalized Butenolides by Oxidative Furan Fragmentation

The development of new chemical transformations to simplify the synthesis of valuable building blocks is a challenging task in organic chemistry and has been the focus of considerable research effort. From a synthetic perspective, it would be ideal if the natural reactivities of feedstock chemicals could be diverted to the production of high value-added compounds which are otherwise tedious to prepare. Here we report a chemical transformation that enables facile and modular synthesis of synthetically challenging yet biologically important functionalized butenolides from easily accessible furans. Specifically, Diels–Alder reactions between furans and singlet oxygen generate versatile hydroperoxide intermediates, which undergo iron(II)-mediated radical fragmentation in the presence of Cu(OAc)2 or various radical trapping reagents to afford butenolides bearing a wide variety of appended remote functional groups, including olefins, halides, azides and aldehydes. The practical utility of this transformation is demonstrated by easy diversification of the products by means of cross-coupling reactions and, most importantly, by its ability to simplify the syntheses of known building blocks of eight biologically active natural products

Titanium-Mediated Dehydroxylative Cross-Coupling of Allylic Alcohols with Electron-Deficient Olefins

Herein we report a Ti(III)-mediated dehydroxylative cross-coupling reaction of allylic alcohols with electron-deficient olefins. This reaction is amenable to various synthetically versatile allylic alcohols, including geraniol and farnesol, providing a general method for dehydroxylative C–C bond formation. We demonstrated the reaction’s utility by simplifying the syntheses of eight useful building blocks that are otherwise laborious to prepare

Advanced Design and Fabrication of Dual-Material Honeycombs for Improved Stiffness and Resilience

Auxetic re-entrant honeycomb (AREH) structures, consisting of a single soft or tough material, have long faced the challenge of balancing stiffness and rebound resilience. To achieve this balance, dual-material printing technology is employed to enhance shock absorption by combining layers of soft and tough materials. Additionally, a novel structure called the curved re-entrant honeycomb (CREH) structure has been introduced to improve stiffness. The selected materials for processing the composite structures of AREH and CREH are the rigid thermoplastic polymer polylactic acid (PLA) and the soft rubber material thermoplastic polyurethane (TPU), created utilizing fused deposition modeling (FDM) 3D printing technology. The influence of the material system and structure type on stress distribution and mechanical response was subsequently investigated. The results revealed that the dual-material printed structures demonstrated later entry into the densification phase compared to the single-material printed structures. Moreover, the soft material in the interlayer offered exceptional protection, thereby ensuring the overall integrity of the structure. These findings effectively serve as a reference for the design of dual-material re-entrant honeycombs

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Additional file 6: Table S2. Plasmids used in this study

Additional file 1: of A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63

Includes the supplementary Figures S1–4. Figure S1. BAF is essential for epidermal gene induction. (a) Quantitative RT-PCR analysis demonstrating the knockdown efficiency of BRG1/BRM siRNAs and the suppression of differentiation gene expression in BAF loss compared to control. (b) GO analysis of the significant changed genes (fold change > 3, FDR < 0.01) with BAF knockdown in RNA-seq. (c) Western blot analysis showing the time course of 4-day differentiation induction in primary human keratinocytes, comparing BRG1/BRM loss with control. BRG1/BRM siRNA efficiently knocked down BRG1 and BRM protein levels. The induction of Keratin 1 is significantly impaired with BAF loss, although no significant changes were detected with p63 and p53 protein level relative to tubulin loading control. (d–f) Distribution of total ATAC-seq peaks, and the BAF-dependent ATAC-seq peaks relative to gene promoter, exon, intron and distal regulatory regions. Figure S2. BAF maintains open chromatin regions with p63 binding sites. (a) Scatter plot demonstrating correlation between BAF binding and open chromatin across the genome. (b) RNA expression levels (RPKM) of p53 family transcription factors in human keratinocytes. (c) Pie chart demonstrating the percentage of p63 motif sites at p63 binding sites that became inaccessible with BAF knockdown. (d) RNA expression levels of all expressed TFs in keratinocytes. Gray dots indicate the expression levels of 809 TFs (RPKM > 1 in differentiating human keratinocytes) listed in GO. Representative TFs known to be functional in epidermal differentiation along with CTCF are highlighted in brown and red. (e) ATAC-seq accessibility in KLF4 motif sites in KLF4 binding sites comparing control vs BAF loss conditions. (f) Heatmap showing the fold changes of the shared 236 genes (fold change > 3, FDR < 0.01) controlled by both BAF and p63. (g) Representative RNA-seq data tracks of BAFi, p63i, and CTRLi replicates. Figure S3. BAF loss does not affect nucleosome positioning or genome accessibility at CTCF binding regions. (a) ATAC-seq fragment size distribution. Gray shaded area represents nucleosome-free fragments (<100 bp), and blue shaded area represents mononucleosome fragments (180–247 bp). Schematic illustration of these ATAC-seq fragments is shown on the right. (b, c) V-plot analysis demonstrating the nucleosome positioning at CTCF motif regions comparing control versus BAF loss. (d) Average diagram of nucleosome-free ATAC-seq fragments at CTCF motif regions comparing control and BAF loss. (e) Average diagram of mononucleosome ATAC-seq fragments at CTCF motif regions comparing control and BAF loss. (f) Average diagram of predicted nucleosome binding probability based on DNA sequences using same number of shuffled genomic regions as in p63 motif nucleosome probability analysis. Figure S4. BAF loss impairs p63 binding to its target sites. (a, b) Single nucleotide ATAC accessibility analysis with 1-bp resolution at p63 and CTCF motif regions in their ChIP-seq binding sites, comparing control and BAF loss. (c) Summit-centered heatmap comparing p63 ChIP-seq peaks in control and BAF loss. (d, e) Average diagram of p63 ChIP-seq signal enrichment in the peaks that are overlapped or unique in control. (f) Average diagram of BAF ChIP-seq signal at CTCF sites comparing p63 loss with control conditions. (PDF 3349 kb

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Additional file 5: Table S1. Primers used in this study

Additional file 2: Table S1. of A novel ATAC-seq approach reveals lineage-specific reinforcement of the open chromatin landscape via cooperation between BAF and p63

Genes changed >3-fold with BRG1/BRM knockdown. Table S2 Shared genes controlled by both BAF and p63 with fold change > 3. Table S3 Sequencing depth of the ATAC-seq, RNA-seq, and ChIP-seq data generated for this study. (XLSX 78 kb