E coli bl21 de3

The expression vectors pUCBAD-MBP-OGT and pET-28a(+)-ESRRB were individually transformed or co-transformed in E.coli BL21 (DE3) (Transgen) and the recombination expression of OGT or/and ESRRB were induced with 0.5% arabinose (wt/vol) or/and 0.5 mM IPTG at 25 °C for 16 h in a shaker as reported^{39 (link)}.

The pEASY-T3 plasmid and the Escherichia coli Trans1 strain (Transgen, Beijing, China) were used for gene cloning and plasmid propagation throughout the study. The pET-28a(+) plasmid (Merck, Darmstadt, Germany) and E. coli BL21(DE3) (Transgen, Beijing, China) were used for recombinant enzyme expression. The genomic DNA of C. bescii DSMZ 6725 was purchased from the Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (https://www.dsmz.de/).

pET-28a (+) expression kit, pEASY-T1 cloning plasmid, RT-PCR kit, E. coli BL21(DE3), ProteinIso Ni-IDA Resin, and E. coli DH 5α were from Transgen Biotech. The gel imaging system, SDS-PAGE, PCR amplification, and electrophoresis devices were manufactured by Bio-Rad Company (United States). Chirascan qCD was manufactured by Applied Photophysics Ltd. (United Kingdom). Plasmid Cas9-NAT carrying nourseothricin and ampicillin resistance genes was obtained from Addgene. The S. cerevisiae HXK2 guide RNA (gRNA) expression vector (HXK2-gRNA) for the expression of the 20 bp gRNA was obtained through the amplification of the gRNA-Trp-Hyb plasmid (Addgene) using the designed primers in Table 1. A. flavus AFB₁ was from FERMENTEK (Israel). Primer synthesis and gene sequencing were performed by Sangon Company (China). The analytically pure reagents were from Aladdin Reagent Company (China).

The gene encoding leaf-branch compost cutinase (LCC, GenBank: AEV21261), TrCBM (GenBank: AGI55989.1) and CfCBM (GenBank: P07984.1) were synthesized by Sangon Biotech (Shanghai, China) and the gene sequences of fusion proteins were listed in Table S7. E. coli BL21(DE3) and DH5α were purchased from TransGen Biotech (Beijing, China). The pET-26b(+) plasmid was obtained from GenScript (Nanjing, China). Ni²⁺-Sepharose 6FF was purchased from Solarbio (Beijing, China). Q5 high-fidelity 2× master mix^®, DpnI and T4 DNA ligase were purchased from New England Biolabs (Beijing, China). Yeast extract, casein tryptone, agar, kanamycin, ampicillin, PET granules, BHET, MHET and TPA were obtained from Sigma-Aldrich (Shanghai, China).

E. coli BL21(DE3), used as a host for the heterologous expression of xylanase, was purchased from TransGen Biotech (Beijing, China). Luria–Bertani (LB) medium was used to cultivate E. coli BL21(DE3) at 37°C. High-affinity Ni-NTA resins (L00250-C) were bought from GenScript (Nanjing, China). Xylans from bagasse, beechwood, birchwood, corncob and oat-spelt, as well as sodium carboxymethyl cellulose (CMC) and Avicel, were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade and commercially available.

The full-length cDNAs encoding the mature Dnm1, Dnm2, and Dnm3 were amplified by the primers (Dnm1-ex-F: 5′-TATCGGATCCGAATTCCTGCAAGATGCATTTACACAG-3′, Dnm1-ex-R: 5′-GGTGGTGGTGCTCGAGATGTGGGCGCTGAGGCACTTGA-3′; Dnm2-ex-F: 5′-TATCGGATCCGAATTCCAGGATGTCTTCAACACCGTC-3′, Dnm2-ex-R: 5′-GGTGGTGGTGCTCGAGGATTTCACCAATAATCACGTTAGC-3′; and Dnm3-ex-F: 5′-TATCGGATCCGAATTCGGAGTTAGCCAGTCACGGTG-3′, Dnm3-ex-R: 5′-GGTGGTGGTGCTCGAGTGACAGAGCATCTTCCCACTG-3′), with EcoRI and XhoI sites in the forward and reverse primers, respectively. The amplified products were cloned into pET30a(+) vector (Novagen) and transformed into competent cells of E. coli BL21 (DE3) (TransGen Biotech, Beijing, China). The positive clones were screened by PCR with specific primers and confirmed by further nucleotide sequencing. The cells were grown at 37 °C in LB medium under agitation until they reached an OD_{600 nm} of 0.6–0.8. Recombinant expression was induced by the addition of isopropyl-b-d-1-thiogalactopyranoside (IPTG; 0.5 mmol/L), followed by incubation for another 4 h under the same conditions. The recombinant rDnm1, rDnm2, and rDnm3 proteins were separated by reducing 12.5% SDS polyacrylamide gel electrophoresis (SDS–PAGE) and visualized with Coomassie brilliant blue R250. rDnm1, rDnm2, and rDnm3 with a His tag were purified by His Bind resin chromatography (Novagen, USA) according to the manufacturer’s instructions.