(1) BAR1 Gene and Effector Protein Expression: 

In this study, we employed CRISPR/dCas technology to construct a logic genetic switch. This device features a stringent conditional triggering mechanism: downstream target gene expression is activated only when three necessary conditions are simultaneously met (detection of high postprandial intestinal glucose concentration + the presence of copper ions in the human gut + the specific hypoxic environment of the intestines). Downstream of these three condition-specific promoters are linked the three essential components required for the CRISPR/dCas9 system: the dCas binding protein,the guide RNA,and the repressor domain MXI.

We plan to design a corresponding scRNA targeting the upstream region of BAR1.Only when all three components are co-expressed will they assemble and bind to the constitutive promoter PBAR1, thereby inhibiting the expression of the BAR1 gene.As a result, α-factor is not degraded (α-factor is a crucial component of the quorum sensing system), allowing the quorum sensing system to proceed normally.

Fig 3. Expression of the BAR1 gene & effector proteins

(2) Quorum Sensing-Controlled Degradation Enzyme Expression: 

To prevent premature enzyme expression from affecting bacterial growth and reducing efficiency, we incorporated a quorum sensing system to monitor its growth status. Only when the engineered bacteria reach a certain density within the intestine and the upstream conditions are met (presence of α-factor), the FUS1 promoter controlling enzyme expression is activated. Subsequently,the downstream PET-degrading enzyme and MHET-degrading enzyme are expressed and,guided by the α-factor signal peptide, secreted outside the engineered bacteria.This leads to further amplification of expression,enabling efficient degradation of microplastics.

Fig 4. Degradation enzyme expression controlled by group sensing

Based on literature, we learned that the adsorption of microplastics can enhance the efficacy of enzymatic degradation^[9][10]. Since hydrophobin HFBI demonstrates high adsorption affinity for PET microplastics^[11], we utilized the α-agglutinin system of Saccharomyces cerevisiae to construct a surface display system for the constitutive expression of HFBI. AGA1 and AGA2 form a complex linked by a disulfide bond intracellularly, and are subsequently displayed on the cell surface via the Aga2sp signal peptide. The V5 epitope tag was incorporated to enable validation of expression without interfering with HFBI's function. Consequently, the engineered Saccharomyces cerevisiae gains the capability to adhere to the surface of PET materials.

Fig 5. The absorption mechanism of the yeast

Furthermore, we noted that in Saccharomyces cerevisiae with gal80 knockout, the use of the gal1,10 promoter enables dynamic regulation of gene expression. This system allows for inducible expression under low glucose conditions—such as during non-feeding periods—which operates orthogonally to the degradation module. The degradation module is activated during feeding, i.e., under high glucose conditions. This temporal separation in the expression of the two modules helps reduce the metabolic burden on the yeast cells, thereby minimizing potential cytotoxicity.

Fig 6. Dynamic regulation mechanism of yeast

We designed a multigene co-expression cassette driven by the GPX2 promoter, constructing an integrated functional module for "antioxidation–mucosal repair–oxidative clearance". The cassette structure is GPX2→NCW2-SOD1→P2A→NCW2-TFF3→T2A→NCW2-CTT1. This design utilizes 2A peptides to enable the continuous expression and self-cleavage of the three functional proteins. An NCW2 signal peptide is introduced before each protein to direct their extracellular secretion, ensuring efficient release and function of the proteins in the intestinal environment. Within this system, SOD1 serves as the first line of defense by scavenging superoxide anions, TFF3 promotes intestinal mucosal repair, and CTT1 decomposes hydrogen peroxide. Together with SOD1, they form a cascade reaction that synergistically clears oxidative stress products, thereby achieving an integrated protective effect of response, repair, and clearance in the gut.

Fig 7. Multi-gene co-expression circuit

Fig 8. The symbiotic system of Saccharomyces cerevisiae and Lactobacillus plantarum

Fig 9. Lactobacillus plantarum co-aggregate with S.cerevisiae through Msa protein

Building upon the adsorption module, the engineered yeast surface-displays hydrophobin HFBI, enabling active adsorption of microplastics. Simultaneously, Lactiplantibacillus strains also adsorb microplastics and, through the mediation of the Msa protein, co-aggregate with the yeast to form a biofilm. This structure facilitates the establishment and maintenance of a stable symbiotic system.

Fig 10. Metabolic complementary symbiotic mechanisms between S.cerevisiae and LAB

This figure illustrates the mechanism of metabolic cross-feeding and symbiosis between Saccharomyces cerevisiae and Lactiplantibacillus. In this system, Lactiplantibacillus breaks down exogenous lactose, releasing galactose as a carbon source for the yeast. Concurrently, metabolic byproducts such as lactic acid or acetic acid can induce the yeast to reduce ethanol production, thereby creating a more favorable environment for Lactiplantibacillus. In return, the yeast secretes growth factors—including amino acids, riboflavin, and CO₂—via nitrogen overflow, which support the growth of Lactiplantibacillus. Additionally, under bile salt-induced stress, the yeast produces a reactivation factor that exhibits protective effects on Lactiplantibacillus.Collectively, this symbiotic system demonstrates immunomodulatory capabilities characterized by both pro- and anti-inflammatory effects, contributing to the alleviation of microplastic-induced intestinal inflammation.

Lactobacillus can bind to adhesion sites on the human intestinal epithelium, mediating colonization.

We discovered a strain called Lactobacillus plantarum DT88, which not only has the potential to co-aggregate with yeast but also exhibits a strong ability to adsorb microplastics on its own.

(1) In Vivo Biosafety — L-Arabinose

Toxin/Antitoxin System:The Endogenous K1 Killer Toxin System in Saccharomyces cerevisiae.This system is naturally present in certain yeast strains and is encoded by viral dsRNA. The K1 killer precursor protein functions as an antitoxin, counteracting the toxicity of the mature K1 killer toxin. We utilized a common food additive—arabinose—to achieve in vivo safety control. In the absence of arabinose in the gut, the K1 killer precursor protein is induced and expressed, providing antitoxin activity. Conversely, upon arabinose intake, the mature K1 killer toxin accumulates, depletes the antitoxin, and ultimately induces cell death.

Fig 11. The suicide mechanism of the yeast

(2) Ex Vivo Biosafety

Due to the temperature difference between the internal and external environments, we employed a temperature-sensitive promoter to regulate an external safety circuit. The HSP26 promoter is induced at temperatures above 37°C, leading to the expression of tetR, which represses the Ptet promoter and prevents the activation of the downstream killer module. Once the engineered yeast is excreted into the external environment where the temperature is insufficient for induction, the killer module is expressed.

For the killer module, we targeted nucleic acid metabolism. First, we identified a core gene in yeast nucleic acid metabolism that encodes phosphoribosylaminoimidazole carboxylase, a key enzyme in the de novo purine biosynthesis pathway. Knocking out ADE2 results in an adenine auxotrophic yeast strain that cannot survive in adenine-deficient environments^[12].

Fig 12. ADE2-regulated suicide mechanism

Second, we introduced a heterologous nuclease gene, nucA, which has been previously demonstrated to function in Saccharomyces cerevisiae^[13]. We placed the nucA gene from Serratia marcescens under the control of the glucose-repressible ADH2 promoter. In the glucose-rich intestinal environment, the ADH2 promoter remains repressed, and nucA is not expressed, allowing the yeast to survive normally. However, if the engineered bacteria escape into external environments with scarce glucose, such as soil or water, the ADH2 promoter becomes activated. This leads to the production of nuclease, which degrades the host cell's genetic material and causes cell death.

Fig 13. NucA-regulated suicide mechanism