Anhydrotetracycline-Inducible Metabolic Flux Adaptation for 3-Hydroxypropionic Acid Production in Pichia pastoris

Microbial cell factories are vital for the sustainable production of chemicals, fuels, and pharmaceuticals, yet their performance depends on the precise control of gene expression to balance metabolic flux between cell growth and production. While the regulation of metabolic networks through constitutive gene expression can promote high-level production, it also imposes a metabolic burden that adversely affects cellular growth. Here, we developed a versatile and high-efficiency inducible platform for the methylotrophic yeast Pichia pastoris, which enables dynamic gene expression for 3-hydroxypropionic acid (3-HP) production from sole methanol. We first constructed an anhydrotetracycline (aTc) inducible promoter library of diverse strengths modified from the constitutive promoter P_GCW14. The promoter library consisted of 32 inducible promoters with the activated strengths ranging from 2.5% to 84.7% of P_GCW14. Second, we dynamically regulated key genes in the 3-HP biosynthesis pathway by aTc inducible promoters, including malonyl-CoA reductase gene (MCR) from Chloroflexus aurantiacus with strong promoters RM5 and RM6, endogenous malonyl-CoA carboxylase gene ACC1 with the very strong promoter RM1, and transmembrane permease PpMFS with the medium promoter RM12. The final engineered strain SHP18 produced 3.7 g/L 3-HP in a shake flask by using methanol as the sole carbon source. This modular aTc inducible promoter toolkit provided finely graded transcriptional control in P. pastoris, which offers a broadly applicable strategy for cell factory design.