Escherichia coli top10

Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. To carry out rapid amplification of cDNA ends (5′ and 3′-RACE), two specific primers (TEGSP5 and TEGSP3, Table S1) were designed based on the tesk1 partial cDNA sequence from tongue sole genome sequencing. We conducted 5′-RACE and 3′-RACE using the SMART RACE cDNA Amplification Kit (Clontech Inc., Mountain View, CA, USA) following the manufacturer's instructions. Touchdown PCR was performed as follows: five cycles of 94°C for 30 s and 72°C for 3 min; five cycles of 94°C for 30 s, 70°C for 30 s, and 72°C for 3 min; 25 cycles of 94°C for 30 s, 68°C for 30 s, and 72°C for 3 min; then 72°C for 10 min as an elongation. The amplified products were electrophoresed on a 1% agarose gel. Bands of expected size were excised from the gel and purified with a Zymoclean Gel DNA Recovery Kit (ZYMO Research, Orange, CA, USA). Purified fragments were cloned into a pMD18-T vector (TaKaRa, Dalian, China), propagated in Escherichia Coli TOP10 (Tiangen, Beijing, China), and positive clones were sequenced on an ABI 3730xl DNA analyzer (Applied Biosystems, Foster City, CA, USA) using SP6 or T7 primers.

Beech wood xylan (BWX, Sigma, USA), oat-spelt xylan (OSX, Shanghai Hualan Chemical Technology Co., Ltd, China), xylooligosaccharides (XOS, Longlive Bio-Technology Co., China), Avicel (Kepujia Reagent Co., China), corncob particles, steam explosion pretreated corn straw (SETCS), grinded corn straw and grinded rice straw were used for hydrolysis experiments. SETCS was provided by Chen lab from Institute of Process Engineering, Chinese Academy of Sciences. Corncob particles, grinded corn straw and grinded rice straw were products treated by mechanical processing by pulverizer (400Y, Kepujia Reagent Co., China). The xylan content (w/w, %) of rice straw, corn straw and corncob particles and SETCS are 22, 17, 36, and 0%. Escherichia coli Top10 (TianGen, China) was used for gene cloning and plasmid maintenance. E. coli BL21 (DE3) and Rosetta (DE3) were applied for gene expression. Plasmid pET-28b (+) (Novagen, USA) was used for recombinant plasmid construction and gene expression. C. kronotskyensis 2002 (DSM 18902) was purchased from DSMZ (Braunschweig, Germany). All of the other reagents and chemicals stated were of analytical grade.

The extremely thermophilic cellulolytic bacteria C. owensensis DSM 13,100 was purchased from the DSMZ (German Collection of Microorganisms and Cell Cultures). The substrates, p-nitrophenyl β-d-galactopyranoside (pNPGal), p-nitrophenyl β-d-glucopyranoside (pNPGlu), p-nitrophenyl β-d-cellobioside (pNPC), p-nitrophenyl β-d-xylopyranoside (pNPX), p-nitrophenyl β-d-mannopyranoside (pNPM), p-nitrophenyl α-l-arabinofuranoside (pNPAr), carboxymethylcellulose (CMC), locust bean gum, and synanthrin, were purchased from Sigma. The chemicals and other substrates were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd or Sigma. The steam-exploded corn straw was obtained by being pretreated in the condition of 1.5 MPa retained for 3 min. Its composition was glucan 46.8 %, xylan 4.3 %, araban 0 %, and lignin 27.4 %. Competent cells used for cloning and expression were Escherichia coli Top10 (TianGen, China) and E. coli BL21 (DE3), respectively. The E.coli BL21 (DE3) competent cells were prepared with the methods described in Molecular Cloning: A Laboratory Manual [43 ].