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Luo et al. Microbiome Res Rep 2025;4:10 https://dx.doi.org/10.20517/mrr.2024.57 Page 11 of 25

Figure 3. Schematic of the working principle of a biosensor constructed using engineered bacteria.

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through pigment production . Li et al. expanded on this concept by engineering Lactobacillus plantarum
WCSF I with an agrQS system for detecting AIP-I. Initially employing GFP as a reporter for fluorescence-
based detection, they subsequently modified the system to secrete lysozyme upon AIP-I detection, creating a
dual-function biosensor that detects and inhibits S. aureus growth .
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Additionally, Mao et al. engineered Lactococcus lactis to detect Vibrio cholerae using a hybrid receptor HR
capable of sensing V. cholerae’s population sensing molecule CAI-1. They utilized fluorescent and
colorimetric reporter genes such as mCherry and β-lactamase for in vitro and in vivo detection, showcasing
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the versatility of engineered bacteria in diverse detection environments . While the sex pheromone of
certain Gram-negative bacteria can serve as a detection signal, Borrero et al. utilized the Enterococci sex
pheromone, cCF10, to construct a pCF10-based expression vector introduced into Lactococcus lactis
NZ9000 for detecting Enterococci presence . The development of these biosensors illustrates the
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significant potential of engineered Lactococcus lactis in pathogenic bacteria monitoring.

It is evident that mere detection of pathogenic bacteria is insufficient; there is a desire to extend biosensor
capabilities to detect biomarkers that hold greater significance in disease diagnosis. Biomarkers encompass
various characteristics (such as cells, proteins, molecules, and genes) measurable from biological samples.
Their presence or fluctuation can indicate the status or progression of specific diseases, playing pivotal roles
in disease prevention, diagnosis, treatment, detection, and prognosis assessment . This expectation has
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been partially realized in biosensors utilizing probiotics as chassis, exemplified by Mimee et al. They
integrated hemoglobin-sensing, genetically modified EcN with ultra-low-power microelectronics to develop
an ingestible micro-bio-electronic system (IMBED). This system detects luminescence signals from bacteria
and wirelessly transmits them to external devices, effectively detecting gastrointestinal hemorrhage in
mouse and pig models . In the context of gut diseases like IBD, genetically engineered EcN, known as i-
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ROBOT, has been pivotal. Leveraging CRISPR-Cas9 gene editing technology, i-ROBOT detects
inflammatory marker thiosulfate levels, expressing associated fluorescent proteins to diagnose IBD
sensitively . Similarly, calreticulin serves as another non-invasive IBD marker . For tumor diagnostics,
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engineered EcN expressing tumor biomarker nitroreductase (NTR) has shown promise. Exploiting its
natural tumor tropism, EcN stably expresses NTR in tumor tissues, activating fluorescent probes for tumor
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imaging, thereby aiding in tumor diagnosis and surgical resection . Similarly, EcN was engineered to
express β-galactosidase in tumor cells, which subsequently degrades the injected substrate LuGal, resulting
in the production of fluorescein detectable in the urine . These studies have demonstrated non-invasive,
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