Inducible Synthetic Growth Regulation Using the ClpXP Proteasome Enhances cis,cis-Muconic Acid and Glycolic Acid Yields in Saccharomyces cerevisiae

Abstract

Engineered microbial cells can produce sustainable chemistry, but the production competes for resources with growth. Inducible synthetic control over the resource use would enable fast accumulation of sufficient biomass and then divert the resources to production. We developed inducible synthetic resource-use control overSaccharomyces cerevisiae by expressing a bacterial ClpXP proteasome from an inducible promoter. By individually targeting growth-essential metabolic enzymes Aro1, Hom3, and Acc1 to the ClpXP proteasome, cell growth could be efficiently repressed during cultivation. The ClpXP proteasome was specific to the target proteins, and there was no reduction in the targets when ClpXP was not induced. The inducible growth repression improved product yields from glucose (cis,cis-muconic acid) and per biomass (cis,cis-muconic acid and glycolic acid). The inducible ClpXP proteasome tackles uncertainties in strain optimization by enabling model-guided repression of competing, growth-essential, and metabolic enzymes. Most importantly, it allows improving production without compromising biomass accumulation when uninduced; therefore, it is expected to mitigate strain stability and low productivity challenges.

Recent Advances in Muconic Acid Extraction Process

“…Numerous attempts have been made to develop a biotechnological technique for the synthesis of muconic acid that is economical, environmentally benign, and sustainable [1,6]. Studies using the strains of E. coli [3], Saccharomyces cerevisiae [7][8][9][10], Pseudomonas [5,11], and Pichia occidentalis [1] have been reported on MA production from different Cis,cis-muconic acid is a well-known platform chemical derived from the catechol catabolic pathway that can be produced from aromatic molecules derived from lignin, carbohydrates, and waste plastics [5]. It can be obtained through chemical synthesis, starting from pricey catechol as raw material, but this method requires environmentally sensitive, non-renewable petroleum-based feedstock and large concentrations of heavy metal catalysts, and the final product is a mixture of two isomers (cis,cis-MA and cis,trans-MA), [1].…”

“…Numerous attempts have been made to develop a biotechnological technique for the synthesis of muconic acid that is economical, environmentally benign, and sustainable [1,6]. Studies using the strains of E. coli [3], Saccharomyces cerevisiae [7][8][9][10], Pseudomonas [5,11], and Pichia occidentalis [1] have been reported on MA production from different kinds of carbon sources. Phosphoenolpyruvate and erythrose 4-phosphate, precursors of MA Appl.…”