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

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.经过工程改造的微生物细胞可合成可持续化学品，但产物合成过程会与细胞生长竞争有限资源。对资源利用实施可诱导的合成型调控，可使细胞快速积累足量生物量，随后将资源转向产物合成路径。本研究通过可诱导启动子表达细菌ClpXP蛋白酶体，在酿酒酵母（Saccharomyces cerevisiae）中构建了可诱导的合成型资源利用调控系统。通过将生长必需的代谢酶Aro1、Hom3与Acc1分别靶向递送至ClpXP蛋白酶体，可在培养过程中高效抑制细胞生长。ClpXP蛋白酶体对靶标蛋白具有严格特异性，未诱导ClpXP表达时，靶标蛋白水平无显著降低。该可诱导生长抑制策略提升了葡萄糖底物的产物得率（目标产物为顺,顺-粘康酸）以及单位生物量的产物得率（目标产物为顺,顺-粘康酸与乙醇酸）。该可诱导ClpXP蛋白酶体系统可通过模型引导的方式，抑制与生长竞争的生长必需代谢酶，从而解决菌株优化过程中的不确定性挑战。尤为关键的是，该系统可在未诱导状态下提升产物合成能力且不影响生物量积累，因此有望缓解菌株稳定性不佳与产物生产力低下的行业难题。