Strains, plasmids, and media
All strains and plasmids used in this study are listed in Table
S1 . The variants were constructed by whole plasmid PCR protocol with
the main primers listed in Table S2 . The PCR system (100 µl)
was composed of template (100‐150 ng), corresponding primers (20 µM with
2 µl), PrimeSTAR polymerase (1 µl; Takara Biomedical Technology, Dalian,
China), 5 × PrimeSTAR Buffer (20 µl), dNTP mix (8 µl), and sterilized
water. Next, DpnI was added to the PCR reaction mixture and incubated
for 3 h at 37°C to eliminate the template plasmid. The digested product
was transformed into Escherichia coli BL21(DE3) cells for the
following screening or DNA sequencing (Genewiz, China). Luria-Bertani
(LB) medium containing 5 g·L-1 yeast extract, 10
g·L-1 tryptone, and 10 g·L-1 NaCl
was used for strain selection and propagation. Terrific Broth (TB)
medium containing 4 g·L-1 glycerin, 24
g·L-1 yeast extract, 12 g·L-1tryptone, 2.31 g·L-1KH2PO4, and 16.43
g·L-1 K2HPO4 was used
for protein expression. Chloramphenicol (30 μg·mL-1),
kanamycin (50 μg·mL-1), and ampicillin (100
μg·mL-1), and IPTG (0.4 mM) were added at the
appropriate time.