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Page 8 of 25 Luo et al. Microbiome Res Rep 2025;4:10 https://dx.doi.org/10.20517/mrr.2024.57

expression systems is the biosafety concern related to the antibiotic resistance genes they often contain.
Both the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA) have
stated that food-grade vectors must be free of antimicrobial resistance elements. Fortunately, alternative
methods exist to eliminate antibiotic resistance genes, such as gene editing using the λ Red recombination
system. This system typically involves three key proteins: the Exo protein, which degrades single-stranded
DNA; the β protein, which facilitates homologous recombination; and the γ protein, which helps convert
linear DNA into a circular form for incorporation into the chromosome. Isabella et al. selected the genes for
PAL and L-amino acid deaminase (LAAD) as the target genes for Phe degradation and integrated them into
the EcN chromosome using the λ Red system. A special plasmid was then introduced to recognize and
excise the expression cassette containing the antibiotic resistance genes, using flippase (FLP) recombinase.
The expression of the PAL and LAAD genes was controlled by the hypoxia-inducible promoter PfnrS,
allowing for gene expression in the hypoxic conditions of the intestine. The engineered bacterium,
SYNB1618, was shown to reduce Phe levels in mice and rhesus monkeys by converting Phe into trans-
cinnamic acid, which is subsequently metabolized into uric acid and excreted by the host .
[74]
[75]
Several engineered bacterial strains, including TYS8500 , SYNB1618 [74,76] , and SYNB1934 [77,78] , utilize EcN as
a vector to express PAL effectively, thereby improving Phe degradation rates. SYNB1618 demonstrated
11
safety and tolerability in clinical trials, achieving a maximum tolerated dose of 2 × 10 colony-forming
[76]
units . SYNB1934, optimized for PAL enzyme activity through directed evolution, exhibited enhanced
enzyme efficacy and stability, alongside favorable safety and pharmacokinetic profiles in non-human
[78]
primate models . These studies propose innovative approaches for PKU treatment using engineered
bacteria as a therapeutic strategy.

Cancer
Historically, tumor regression has been observed in association with local infections . Advances in
[79]
understanding the human microbiome, particularly the concept of intratumoral microbiota, highlight the
critical role of bacteria in tumor growth. Previous studies have shown that certain bacteria can target and
colonize tumor tissue, playing a role in the formation of the tumor microenvironment. For example, EcN,
attenuated Salmonella typhimurium, and Lactobacillus paracasei have demonstrated this ability [80-82] . Tumor
colonization can occur passively, such as when vascular disruption, caused by a sudden increase in TNF-α
[83]
in the tumor vasculature, leads to bacterial influx . Additionally, Salmonella typhimurium strains lacking
certain chemotactic receptors (e.g., tar, tsr, trg receptors) lose their ability to colonize tumors, suggesting
that specific chemical signals in tumor tissues may guide bacterial colonization . Furthermore, the low-
[84]
oxygen environment of tumor tissues may attract anaerobic bacteria, supporting their colonization. Bacteria
colonizing tumor tissues can enhance the body’s ability to fight tumors by reprogramming the tumor
microenvironment, particularly through the modulation of immune cells . For example, Lactococcus lactis
[85]
subsp. cremoris C60 induces a macrophage inflammatory phenotype via TLR signaling, which promotes
+
antigen-dependent activation of tumor-specific CD8 T cells, thereby enhancing the immune response
against melanoma . Additionally, Lactobacillus plantarum L168 and its metabolite indole-3-lactic acid
[86]
+
(ILA) stimulate IL-12 production by dendritic cells (DCs), leading to CD8 T cell activation and improved
outcomes in colorectal cancer.
In summary, the specific mechanisms through which natural bacteria target and colonize tumors remain
under investigation. However, these bacteria are already promising candidates for chassis engineering due
to their potential therapeutic advantages. Before such applications can be realized, the safety concerns
surrounding certain bacteria, such as Salmonella typhimurium, need to be addressed. Salmonella
typhimurium is a Gram-negative, foodborne pathogen that causes gastrointestinal symptoms and systemic