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To ensure efficient delivery of target molecules to lesions, engineered bacteria often require modifications to
their expression plasmids, such as codon optimization and the addition of secretion signal sequences.
Codon optimization involves altering codons in exogenous gene sequences to align with the host cell's
preferred codon usage, thereby enhancing translation efficiency. This strategy has been widely applied in the
design of engineered bacteria . The process relies heavily on bioinformatics in synthetic biology, alongside
[37]
[38]
advances in molecular biology . Another approach to improving the delivery of heterologous proteins by
engineered bacteria is the incorporation of a secretion signal sequence at the N-terminal of the target gene.
For instance, in a study by Hanson et al., the genes encoding two key subunits of IL-27, IL-27p28 and Ebi3,
were codon-optimized and fused by a contiguous sequence, with a secretion signal sequence added at the
N-terminus. The engineered Lactococcus lactis (LL-IL-27) exhibited enhanced IL-27 delivery capacity. This
modification made LL-IL-27 more effective in treating IBD in mice compared to both LL-IL-10 and
systemically administered recombinant IL-27 [Figure 2]. LL-IL-27 showed efficacy by stimulating IL-10
+
production in T cells, reducing CD4 T cells among intestinal lymphocytes, and mitigating inflammatory
cell infiltration .
[39]
The gastrointestinal environment in IBD presents a complex challenge, as even engineered bacteria that are
optimized for safety and production efficiency may be compromised before they reach their target site for
colonization. To enhance the bioavailability of these bacteria, researchers have combined them with various
biomaterials to create effective drug delivery systems. For example, in a study focused on removing ROS
from the IBD gut, researchers used two biocompatible and biodegradable materials - chitosan and sodium
alginate - to coat engineered EcN expressing catalase (CAT) and superoxide dismutase (SOD) via a layer-
by-layer electrostatic self-assembly technique, resulting in EcN-pE(C/A)2. These biomaterials protected the
engineered bacteria from digestive enzymes and the acidic conditions of the gastrointestinal tract, while also
enhancing their compatibility with other materials. This strategy improved the stability and bioavailability
[40]
of the engineered bacteria . Interestingly, EcN can also be designed as an inactive carrier for drug delivery.
Researchers introduced the lysin E gene from phage φX174 into EcN, inducing its expression to cause the
bacterium to lyse into an empty shell that retains only the surface structure. These “EcN ghosts” have
demonstrated safety and efficacy in IBD treatment .
[41]
To conclude, engineered bacteria function as carriers for localized delivery of live immunoreactive
substances, effectively bypassing systemic symptoms and addressing the short half-life concerns of direct
cytokine delivery. Future advancements in modulating probiotic genes show significant promise for
optimizing this in vivo drug delivery system for treating IBD.

Metabolic disease
Diabetes
Type 2 diabetes (T2D) is characterized by insulin resistance initially, leading to impaired glucose utilization
by tissue cells and eventual pancreatic β-cell dysfunction. Dysregulation of the host’s gut microbiota has
been implicated in T2D development, impacting metabolism, inflammatory responses, and oxidative
stress [42,43] . T2D patients often exhibit an imbalance with increased pathogenic bacteria and reduced butyric
acid-producing bacteria in the gut, influencing host immune and metabolic functions [42,44] . Further clinical
and animal studies have demonstrated that probiotic supplementation with strains such as Lactobacillus
acidophilus, Bifidobacterium lactis, and Lactobacillus casei Shirota can modestly lower blood glucose levels
in T2D patients [45,46] , suggesting a potential role for probiotics in T2D management.

On another front, glucagon-like peptide-1 (GLP-1), a newly discovered gastrointestinal hormone, stimulates
insulin secretion and inhibits glucagon secretion, thereby lowering blood glucose levels [47,48] . However, its