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.