Abstract Microbial cell factories provide a green and sustainable opportunity to produce value‐added products from renewable feedstock. However, the leakage of toxic or volatile intermediates decreases the efficiency of microbial cell factories. In this study, membraneless organelles (MLOs) were reconstructed in Saccharomyces cerevisiae by the disordered protein sequence A‐IDPs. A regulation system was designed to spatiotemporally regulate the size and rigidity of MLOs. Manipulating the MLO size of strain ZP03‐FM, the amounts of assimilated methanol and malate were increased by 162 and 61 , respectively. Furthermore, manipulating the MLO rigidity in strain ZP04‐RB made acetyl‐coA synthesis from oxidative glycolysis change to non‐oxidative glycolysis; consequently, CO2 release decreased by 35 and the n‐butanol yield increased by 20 . This artificial MLO provides a strategy for the co‐localization of enzymes to channel C1 starting materials into value‐added chemicals.
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