Engineered Artificial Membraneless Organelles in Saccharomyces cerevisiae To Enhance Chemical Production

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, CO 2 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 C 1 starting materials into value-added chemicals.

Designing Intracellular Compartments for Efficient Engineered Microbial Cell Factories

“…86,102 It has also been reported that regulating the size and rigidity of synthetic MOLs in Saccharomyces cerevisiae can increase methanol assimilation efficiency and the titer and yield of n-butanol. 103 The combination of protein scaffolds, ligands, lysis, or capacitive proteins and photosensitive proteins to improve the manipulability of ASCs based on LLPS will create considerable prospects for the application of intracellular compartments in MCFs.…”

“…Although the condensation process and degree of spontaneous aggregation of synthetic MOLs cannot be perfectly controlled, recent studies have reported that the aggregation of protein condensates is affected by protein concentration, external membrane environment strength, the presence of RNA, the number of tyrosine–arginine (Y-R) pairs, positive charge, and other factors. , It has also been reported that regulating the size and rigidity of synthetic MOLs in Saccharomyces cerevisiae can increase methanol assimilation efficiency and the titer and yield of n -butanol . The combination of protein scaffolds, ligands, lysis, or capacitive proteins and photosensitive proteins to improve the manipulability of ASCs based on LLPS will create considerable prospects for the application of intracellular compartments in MCFs.…”

A Modular Design for Synthetic Membraneless Organelles Enables Compositional and Functional Control

“…PpID-based functionalization of synthetic condensates thus provides the basis for the continued adoption of membraneless organelles as hubs for synthetic biology 6,23,24 . In particular, recruiting metabolic pathways to condensates can divert flux of intermediates away from competing pathways [30][31][32][33][34] . Indeed, the relative stoichiometry and degree of recruitment of enzymes can significantly impact the resulting product formation 56 .…”

“…For example, we previously introduced the Corelet system, whereby weakly interacting protein domains optogenetically multimerize on spherical protein assemblies of human ferritin, resulting in spatiotemporally tunable phase separation 40 . Others have added interaction domains to recruit various proteins to functionalize related synthetic organelles 26,27,33,34 . However, previous efforts to systematically engineer biomolecular condensates fall short of providing a quantitative framework for controlling the recruitment of various functional proteins into the condensates.…”

Carbon efficient production of chemicals with yeasts

“…Therefore, to overcome the carbon loss in acetyl-CoA synthesis, 2023) also introduced the GATHCYC pathway along with other genetic engineering modifications into a n-butanol producing S. cerevisiae strain that showed an increase in the acetyl-CoA supply that improved the n-butanol titer up to 1.75 g/L with a decrease of 35.2% of the total CO 2 (Zhou et al, 2023).…”