P pastoris x 33

P. oxalicum 114-2 and P. oxalicum RE-10 were stored in our laboratroy. E. coli DH5α and pEASY-Blunt Zero vector from TransGen (Beijing, China) were used for propagation of plasmids and gene clone. The plasmid pPICZαA and host P. pastoris X-33 from Invitrogen (Carlsbad, CA, USA) were employed for the β-xylosidase gene expression. All strains and plasmids used in this study shown in supplementary Table S3.

The yeast host strain P. pastoris X33 and the plasmid pPICZαA were products of Invitrogen (Carlsbad, CA, USA). WCOs (FFA content of 19%, acid value of 170 mg KOH/g, saponification value of 366 mg KOH/g, density of 0.89 g/cm³, water content of 0.1%) were kindly donated by Lvming Environmental Protection Technology Co., Ltd. (Shanghai, China). n-hexadecane and authentic standards of FAMEs were purchased from Sigma-Aldrich (St. Louis, MO). The media including yeast extract peptone dextrose medium (YPDS), BMGY, BMMY, and low-salt lysogeny broth used in this study were prepared by following the manual of EasySelect™ Pichia Expression Kit (Invitrogen).

Strains: P. pastoris X33 (Invitrogen) was cultivated in BMGY medium in the phase of growth phase and BMMY medium in the phase of induction. There is 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base without amino acids, 4 × 10^−5% biotin, and 1% glycerol in BMGY medium. And there is 100 mM potassium phosphate (pH 6.0), 1.34% yeast nitrogen base without amino acids, 4 × 10% biotin, and 0.5% glycerol in BMMY medium. Escherichia coli DH5α were used for plasmid propagation while BL-21(DE3) cells were used for cloning of Lac Z coding sequence. Escherichia coli DH5α was cultivated in LB medium with temperature of 37 °C. And LB medium contains 1% tryptone, 0.5% yeast extract and 0.5% NaCl supplemented with 25 µg ml⁻¹ Zeocin for plasmid maintenance and propagation.
Plasmids: pPICZA was used for constructing mutants of AOX1 promoter while pGAPZαA (Invitrogen) was used for cloning the sequence of GAP promoter for homologous recombination with yeast genome. All plasmids used in this study are listed in Table 1.

The following primers were used to amplify the l-asparaginase gene into P. pastoris X33 strains using PPICZαA vectors by Invitrogen, Carlsbad, CA, USA: Forward primer 5′-ATGCCCAGCTTTAAACGGCTT-3′ and Reverse primer 5-GTGCACTCCCGCGTGCTC-3. Initially, in order to increase the plasmid concentration, E. coli strain TOP 10 were transformed aimed to increase the plasmid concentration. Zeocin (25 μg/mL) for 1 day at 37 °C under 200 rpm. was used in the culture medium for selected the transformed strain—(DNA fragments were purified using the Pure Link Quick Plasmid Miniprep kit (Invitrogen, Carlsbad, CA, USA). The DNA fragments were transformed into P. pastoris X33 strain (Invitrogen, Carlsbad, CA, USA) via electroporation.

The plasmids pPICZα A-wtRBD-co and pPICZα A-Delta RBD-co were linearized with SacI and transformed into the P. pastoris X33 (Invitrogen, Carlsbad) by electroporation (Bio-Rad, Hercules). Yeast clones integrated with wtRBD-co or Delta RBD-co gene were selected on yeast extract peptone dextrose sorbitol (YPDS) plates containing 100 μg/mL zeocin (Invitrogen) at 30°C. Colony PCR was performed using the primer 5’AOX and 3’AOX (Table S2), and the positive clones were selected and grown in 3 mL BMGY medium (Sangon Biotech) at 30°C with a shaking speed of 250 rpm, until the OD600 reached 2.0-6.0. Yeast cells were harvested by centrifugation at 3,000 rpm for 5 min at 4°C, and the pellets were suspended with an equal volume of the BMMY medium (Sangon Biotech). Induction was performed at 30°C with shaking at 250 rpm for 72 h, and additional methanol (0.5% final concentration) was added to the medium every 24 h. The expression levels of RBD protein in the supernatant for individual yeast clones were quantified by western blot analysis.

Microbial strains and plasmids used in this study are listed in Table 1. Enterococcus faecium T136 and Pediococcus damnosus CECT4797 were grown in MRS broth (Oxoid Ltd., Basingstoke, UK) at 32°C. P. pastoris X-33 (Invitrogen S.A., Barcelona, Spain) was cultured in YPD medium (Sigma-Aldrich Inc., St. Louis, MO, USA) at 30°C with shaking (200–250 rpm). Escherichia coli JM109 (Promega, WI, USA) was grown in LB broth (Sigma-Aldrich) at 37°C with shaking (250 rpm). Listeria monocytogenes CECT4032 was grown in LB at 37°C and Salmonella typhimurium CECT443 was grown in TSB (Oxoid) at 37°C. Campylobacter jejuni ATCC33560 and C. jejuni NCTC11168 were grown in BHI supplemented with 1% defibrinated horse serum (BD Bioscience, CA, USA) at 37°C in microaerophilic conditions. E. coli O157:H7 was grown in LB at 37°C with shaking (250 rpm). Yersinia ruckeri LMG3279 was grown in TSB at 28°C. Zeocin (Invitrogen) was added when needed at concentrations of 25, 100, or 1000 μg/mL. Strains cited as CECT belong to the Colección Española de Cultivos Tipo (Valencia, Spain), ATCC to the American Type Culture Collection (Rockville, MD, USA), and NCTC to the National Collection of Type Cultures (London, UK).

Escherichia coli DH5α [genotype: F^- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17 $\mathit{\left(}{\mathit{r}}_{\mathrm{K}}^{\mathrm{-}}{\mathit{m}}_{\mathrm{K}}^{\mathrm{+}}\mathit{\right)}$ ,λ^-] was used as the host strain for cloning and propagation of vector. P. pastoris X-33 (genotype: wild type) (Invitrogen, Carlsbad, CA, USA) was used as the expression host. E. coli clone harboring chimeric α-amylase gene (Ba-Gt-amy) generated earlier (Parashar and Satyanarayana, 2016b (link)) was procured from the laboratory culture collection. pPICZαA vector (Invitrogen) was used as a cloning and expression vector. The restriction enzymes and the primers used in this investigation were purchased from New England Biolabs (Beverly, MA, USA) and Sigma-Aldrich (USA), respectively. Plasmid extraction and gel elution kits were purchased from Real Biotech Corporation (RBC) (Taiwan). Media and other components for trace metal solution used in this experiment are listed in Table 1.