2.2 Genetic constructs and site-directed mutagenesis
The gene coding for Ps XR (GenBank ID: XP_001385181) was
amplified from the genome of Pichia stipites. Genes coding forPd PDH from Paracoccus denitrificans (GenBank ID:
WP_011751060), Rl GDH from Rhizobium leguminosarum(GenBank ID: WP_011650422.1), EutM from Salmonella enterica(GenBank ID: NP_461400.1), and fbaB (SpyTag-Catcher) fromStreptococcus pyogenes (GenBank ID: NZ_LS483338.1) were
synthesized by BGI Genomics Co., Ltd. (Shenzhen, China). All the above
genes were amplified with primers harboring a homologous arm and ligated
into pET-32a to obtain the recombinant vectors pET-32a-Ps XR,
pET-32a-Pd PDH, pET-32a-Rl GDH, and pET-32a-EutM. All
mutants were constructed using pET-32a-Pd PDH as a template for
the KOD-Plus-Mutagenesis Kit. Strains and plasmids used in this study
are listed in Table S1, and primers are summarized in Table S2.