E coli bl21 de3

Briefly, LGG genomic DNA was isolated and used as a template for PCR. Considering the transmembrane structure at the N-terminal of HM0539, primers aimed at 51–357 amino sequences were designed for amplification of extracellular protein fragment:
F: 5′-GTAGAATTCGTTAACGCGGCAACGAAAG
R: 5′-CGGGCCTCGAGTTAGTTGATCACTTCAA.
PCR products were purified using a GenElute^TM PCR Clean-Up Kit (Sigma-Aldrich), and then digested with EcoR I and Xho I, followed by ligating to the same restriction enzyme cutting sites of pET-31a(+) (Novagen). The recombinant plasmid was transformed into E. coli DH5α (Tiangen Biotech Co., Ltd., Beijing, China). After incubation, the kanamycin resistant bacterial colony was lysed for PCR assay. The expression plasmid producing the HM0539 was confirmed by DNA sequencing and transformed into E. coli BL21(DE3) (Tiangen Biotech Co., Ltd., Beijing, China). Recombinant HM0539 protein with a histidine tag was overexpressed via 0.5 mM isopropyl-β-D-thiogalactoside induction at 37°C and purified using Histidine Tagged Protein Purification Kit (Soluble Protein, CW0894, CWBioTech, Beijing, China), according to the manufacturer’s instructions. The potential endotoxin of HM0539 was removed using resin according to the supplier’s instructions (EndotoxinOUT Resin; Sangon Biotech, Shanghai, China).

The primers PF and PR for rTCS was designed and added with NdeI and NheI restriction enzyme sites (Table 1). rTCS gene was amplified from plasmids pbs-rTCS preserved in our laboratory. The DNA fragment was digested with restriction enzymes, and cloned between NdeI and NheI sites of pMD18T-rTCS. The rTCS gene was confirmed by DNA sequencing. The PET22b vector and the rTCS gene were ligated by T4 ligase. The resulted plasmid, named PET22b-rTCS, was transformed into E. coli BL21(DE3) (TIANGEN Biotech Co., Ltd, China) following the standard procedure.Table 1

Primers used for amplification of TCS

Primer	Sequence 5-3	Enzyme site added
Forward	cCATATGatgatcagattcttagtcctc	NdeI
Reverse	gGCTAGCctaaatagcataacttcca	NheI

The expression plasmids pET/entolimod-28a(+), pET/Ssp DnaE/entolimod-28a(+) and pET/Npu DnaE/entolimod-28a(+) were transformed into E. coli BL21(DE3) (Tiangen Biotech, Beijing, China). The cells were grown overnight at 37 °C in LB medium supplemented with 50 μg/mL kanamycin. Protein overexpression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM in the bacterial culture (when optical density at 600 nm reached ∼0.5) and the incubation was extended for additional 3–5 h at 37 °C. The cells containing the expression plasmids pET/entolimod-28a(+), pET/Ssp DnaE/entolimod-28a(+), and pET/Npu DnaE/entolimod-28a(+) were subsequently harvested at 6,000×g for 25 min. After centrifugation, the three cell pellets were respectively sonicated (30% amplitude for 30 min, 5 s on, 5 s off) in lysis buffer: 20 mM Tris, 500 mM NaCl, 10 mM Imidazole, pH 8.0. The target proteins expressed as inclusion bodies (∼80% of total) were confirmed by SDS–PAGE analysis.

In order for the recombinant Mr-IAGBP protein to show phenotypical features, the primers expressed in Table S2 were designed to magnify the full-length ORF of Mr-IAGBP. The PCR products were disinfected and injected into the pMD-19T vector, and subsequently confirmed by sequencing. After, the 1% agarose gel electrophoresis and target products were decontaminated using a TaKaRa Agarose Gel DNA Purification KitVer.2.0 (TaKaRa, Kyoto, Japan). T4 DNA ligase was used to connect the Mr-IAGBP fragment to the empty pET-32a vector and kept overnight at 4 ℃. The expression plasmid pET-32a-Mr-IAGBP was transformed to E.coli BL21(DE3) (TIANGEN, Beijing, China) and then cultured in Amp⁺ LB at 37 °C at a speed of 200 rpm. Isopropyl-β-D-thiogalactopyranpside (IPTG) was added to create a final concentration of 0.5 mmol/L and then cultured at 37 °C at a speed of 200 rpm for 4 h, and then centrifuged at 4 °C at a speed of 12,000 rpm for 10 min. Sediment cells were resuspended in PBS and purified with the His Band Resin columns (Sangon Biotech, Shanghai, China), as per protocol. The concentrations of recombinant pET-32a-Mr-IAGBP protein were determined according to the method specified for the Bradford Protein Assay Kit (Beyotime, Shanghai, China). The purified recombinant protein was separated in a 12% SDS-PAGE gel electrophoresis.

Plasmid pET-30a (+) and E. coli BL21 (DE3) were purchased from Tiangen Biotech Co., Ltd. (Beijing, China). Soluble starch, agarose, carrageen, sodium cellulose, and alginate were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, china). Grateloupia filicina, Chondrus ocellatus, and Scagassum were collected from Yangkou Beach in Qingdao, China.

Thirty of the full-length ORFs were used for heterologous expression in E. coli BL21 (DE3) (Tiangen, Beijing, China) and subsequent cellulase activity assays. Briefly, the above recombinant plasmid containing each of the genes was obtained using a SanPrep Column Plasmid Mini-Preps Kit (Sangon, Shanghai, China) and was transformed into E. coli BL21 (DE3) by heat shock. Transformants were grown in liquid SOC (1 h at 37 °C), plated onto kanamycin (50 µg ml⁻¹)-containing LB agar plates and grown overnight at 37 °C. The colonies confirmed to harbour recombinant plasmid using PCR as described above and stored in liquid cultures [LB, 20% glycerol, kanamycin (50 µg ml⁻¹)] at − 80 °C.

Standards of lycopene (CAS, 502-65-8), β-carotene (CAS, 7235-40-7), zeaxanthin (144-68-3), and crocetin dialdehyde (CAS, 502-70-5) were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO, USA). Phanta Max Super-Fidelity DNA Polymerase was purchased from Vazyme (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). A ClonExpress II One Step Cloning Kit C112 was purchased from Vazyme. IPTG was purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). All restriction endonucleases were purchased from Takara (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). T4 DNA Ligase was purchased from Takara. A DNA plasmid isolation kit and DNA gel extraction kit were purchased from Tiangen (TIANGEN Biotech (Beijing) Co., Ltd., Beijing, China). Tryptone and yeast extract were purchased from Thermo (Thermo Fisher Scientific Inc., Waltham, MA, USA). E. coli DH5α and E. coli BL21 (DE3) were purchased from Tiangen. Z/B/L represent the BL21 (DE3) harboring pACCAR25ΔcrtX, pACCAR16Δcrt, or pACCRT-EIB, respectively. All the chemical reagents utilized in this experiment were of analytical grade.