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to produce a GLP-1-like product locally, exerting beneficial effects in managing diabetes. Researchers have
proposed expressing a cholera toxin B subunit-insulin-like growth factor 1 fusion protein (CTB-IGF-1)
using engineered E. coli Nissle 1917. Here, the cholera toxin B subunit (CTB) serves as a carrier protein
facilitating the targeting of insulin-like growth factor 1 (IGF-1), which mimics insulin's physiological
function via distinct receptors . While this hypothesis has not been confirmed by subsequent reports,
[55]
analogous studies have been conducted. For instance, engineered E. coli BL21 expressing a CTB-10×rolGLP-
1 fusion protein significantly lowered blood glucose levels in mice with T2D . Moreover, engineered E. coli
[56]
BL21 (DE3) expressing β-cell expansion factor A (BefA) holds promise for T2D treatment by enhancing
pancreatic β-cell proliferation while mitigating inflammation and apoptosis [57,58] .

Phenylketonuria
Phenylketonuria (PKU) is an inherited metabolic disorder caused by autosomal recessive inheritance,
mainly resulting from a deficiency of phenylalanine hydroxylase (PAH) in patients. Additionally, the PAH-
catalyzed conversion of phenylalanine (Phe) to tyrosine (Tyr) requires tetrahydrobiopterin (BH4) as a
cofactor. Therefore, patients with impaired BH4 metabolism may also develop PKU . This deficiency leads
[59]
[60]
to the excessive accumulation of phenylalanine, which can damage the nervous system . Historically, a
strict low-Phe diet has been the cornerstone of PKU treatment . However, long-term dietary control does
[61]
not alter the underlying nature of the disease. This approach is not only difficult to implement but may also
[63]
lead to other complications . Recently, new treatment protocols such as gene therapy and enzyme
[62]
[64]
replacement therapy have been proposed and validated. Engineered bacteria could potentially enhance
enzyme replacement therapy for PKU.
Enzymes currently under study for treating PKU include phenylalanine lyase (PAL) and PAH, both crucial
for reducing Phe levels via metabolic pathways [64,65] . The Anabaena variabilis phenylalanine ammonia lyase
gene (AvPAL) was optimized and cloned into Lactobacillus reuteri 100-23C. The engineered bacterium
demonstrated a significant reduction in blood Phe levels in mice within 3 to 4 days of administration .
[66]
AvPAL functions by breaking down accumulated Phe into cis-cinnamic acid and ammonia, which is
excreted through normal metabolic pathways. In contrast, PAH can be secreted into the intestines by
engineered Lactobacillus plantarum CM_PUJ411, detected using the fluorescent protein. This strain
operates through two primary mechanisms: secreted PAH catalyzes the conversion of L-phenylalanine (L-
Phe) to L-tyrosine (L-Tyr), which is then excreted via normal metabolic pathways. Additionally, it expresses
a cell-penetrating peptide, the TAT protein transduction domain, which enhances the intestinal uptake
efficiency of recombinant PAH and potentially reduces degradation within the intestinal lumen . The
[67]
construction of the two types of engineered bacteria mentioned above involves two common plasmid-based
expression systems: the constitutive expression system and the inducible expression system. Typically,
plasmid-based expression systems include key elements such as a replicon, a promoter with a ribosome
binding site and regulatory sequences, a terminator, a target gene, and a selection marker. In a previous
project, a plasmid-based expression system was constructed in Anabaena variabilis, where the promoter of
the AvPAL gene was replaced with a high-yield constitutive promoter from Lactobacillus casei (the
erythromycin resistance B gene, ermB), enabling sustained and stable expression of PAL in the host cells.
PAL expression in the host cell does not require additional inducible signals. In contrast, the pSIP503
expression vector carrying the PAH gene features a nisin-inducible promoter, which drives the transcription
of downstream target genes when nisin levels in the environment are elevated. Both constitutive expression
[71]
systems (e.g., pUBU , pPBT-GFP , pNZ2103 ) and inducible systems (e.g., pMSP3535 , pULP3-
[68]
[69]
[70]
[73]
[72]
PLDH , pMY01 ) offer distinct advantages. Constitutive systems enable stable and efficient expression of
therapeutic proteins in complex intestinal environments, while inducible systems are better suited for
industrial-scale recombinant protein production. However, a significant drawback of plasmid-based